Use of OX-2 to suppress an immune response

ABSTRACT

Methods and compositions for inducing immune suppression are disclosed. The methods involve administering an effective amount of an OX-2 protein or a nucleic acid encoding an OX-2 protein. The methods are useful in preventing graft rejection, fetal loss, autoimmune disease, and allergies. Methods and compositions for preventing immune suppression are also disclosed. The methods involve administering an effective amount of an agent that inhibits OX-2.

This application is a continuation of U.S. patent application Ser. No.09/570,367 filed May 5, 2000 (now U.S. Pat. No. 6,338,851), which is acontinuation of international application No. PCT/CA98/01038 filed Nov.6, 1998, which claims priority to provisional application 60/064,764filed Nov. 7, 1997 (now abandoned).

FIELD OF THE INVENTION

The present invention relates to methods and compositions for modulatingan immune response. The invention includes the use of the protein OX-2to suppress an immune response.

BACKGROUND OF THE INVENTION

The immune system protects the body from infectious agents and diseaseand is critical to our survival. However, in certain instances, theimmune system can be the cause of illness. One example is in autoimmunedisease wherein the immune system attacks its own host tissues, in manyinstances causing debilitating illness and sometimes resulting in death.Examples of autoimmune diseases include multiple sclerosis, type 1insulin-dependent diabetes mellitus, lupus erythematosus and arthritis.A second example where the immune system can cause illness is duringtissue or organ transplantation. Except in the cases of geneticallyidentical animals, such as monozygotic twins, tissue and organtransplants are rejected by the recipient's immune system as foreign.The immune reaction against transplants is even more pronounced intransplantation across species or xenotransplantation. A third examplewhere the immune system harms the host is during an allergic reactionwhere the immune system is activated by a generally innocuous antigencausing inflammation and in some cases tissue damage.

In order to inhibit the detrimental immune reactions duringtransplantation, autoimmune disease and allergic reactions,immunosuppressive drugs (such as cyclosporin A, tacrolimas, andcorticosteroids) or antibody therapies (such as anti-T cell antibodies)are generally administered. Unfortunately, these non-specific modes ofimmunosuppression generally have undesirable side effects. For example,cyclosporin may cause decreased renal function, hypertension, toxicityand it must be administered for the life of the patient. Corticosteroidsmay cause decreased resistance to infection, painful arthritis,osteoporosis and cataracts. The anti-T cell antibodies may cause fever,hypertension, diarrhea or sterile meningitis and are quite expensive.

In view of the problems associated with immunosuppression, there hasbeen an interest in developing methods or therapies that induceunresponsiveness or tolerance in the host to a transplant, to “self”tissues in autoimmune disease and to harmless antigens associated withallergies. The inventor has been studying the mechanisms involved intransplant rejection and has developed methods for inducing a state ofantigen-specific immunological tolerance in transplantation. Inparticular, in animal allograft models, the inventor has demonstratedthat graft survival can been increased if the recipient animal is givena pre-transplant infusion via the portal vein of irradiated spleen cellsfrom the donor animal. In contrast, a pre-transplant infusion via thetail vein does not prolong graft survival.

Understanding the molecular mechanisms involved in the induction oftolerance following portal-venous (pv) immunization may lead to thedevelopment of methods of inducing immune tolerance that may be usefulin transplant, autoimmune disease and allergies.

SUMMARY OF THE INVENTION

The present inventor has identified genes that show an increase inexpression following portal venous immunization. One of the genesisolated encodes OX-2, a molecule with previously unknown functionbelonging to the Ig superfamily. The inventor has shown thatadministering antibodies to OX-2 inhibited the graft survival generallyseen following pre-transplant pv immunization. The inventor has alsoshown that there is a negative association between levels of OX-2 andrisk of fetal loss. The inventor has further shown that OX-2 inhibitscytotoxic cells and IL-2 production and induces IL-4 production. All ofthese results demonstrate that OX-2 is involved in immune suppression.

Consequently, broadly stated, the present invention provides a method ofsuppressing an immune system comprising administering an effectiveamount of an OX-2 protein or a nucleic acid sequence encoding an OX-2protein to an animal in need of such treatment.

In one embodiment, the present invention provides a method of inducingimmune tolerance to a transplanted organ or tissue in a recipient animalcomprising administering an effective amount of an OX-2 protein or anucleic acid sequence encoding an OX-2 protein to the recipient animalprior to the transplantation of the organ or tissue.

In another embodiment, the present invention provides a method ofpreventing or inhibiting graft versus host disease in a recipient animalreceiving an organ or tissue transplant comprising administering aneffective amount of an OX-2 protein or a nucleic acid sequence encodingan OX-2 protein to the organ or tissue prior to the transplantation inthe recipient animal.

In yet another embodiment, the present invention provides a method ofpreventing or inhibiting fetal loss comprising administering aneffective amount of an OX-2 protein or a nucleic acid sequence encodingan OX-2 protein to an animal in need thereof.

In a further embodiment, the present invention provides a method ofpreventing or treating an autoimmune disease comprising administering aneffective amount of an OX-2 protein or a nucleic acid sequence encodingan OX-2 protein to an animal having, suspected of having, or susceptibleto having an autoimmune disease.

In yet a further embodiment, the present invention provides a method ofpreventing or treating an allergy comprising administering an effectiveamount of an OX-2 protein or a nucleic add sequence encoding an OX-2protein to an animal having or suspected of having an allergy.

The invention also includes pharmaceutical compositions containing OX-2proteins or nucleic acids encoding OX-2 proteins for use in inducingtolerance in transplantation or autoimmune disease.

The inventor has cloned and sequenced the murine OX-2 gene. Accordingly,the invention also includes an isolated nucleic acid sequence encoding amurine OX-2 gene and having the sequence shown in FIG. 7 andSEQ.ID.NO.:22 and an isolated murine OX-2 protein having the amino acidsequence shown in FIG. 8 and SEQ.ID.NO.:2.

As stated above, OX-2 can be used to induce immune suppression.Consequently, inhibiting OX-2 may also be useful in preventing immunesuppression.

Therefore, in another aspect, the present invention provides a method ofpreventing immune suppression comprising administering an effectiveamount of an agent that inhibits OX-2 to an animal in need thereof. In apreferred embodiment the OX-2 inhibitor is an antibody that binds OX-2or an antisense oligonucleotide that inhibits the expression of OX-2.

In one embodiment, the present invention provides a method of inducingfetal loss comprising administering an effective amount of an agent thatinhibits OX-2 to an animal in need thereof.

The invention also includes pharmaceutical compositions containing anOX-2 inhibitor for use in inducing or augmenting an immune response.

Other features and advantages of the present invention will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings inwhich:

FIG. 1 illustrates PCR validation of suppressive subtractivehybridization using β-actin primers.

FIG. 2 illustrates PCR validation of suppressive subtractivehybridization using IL-10 primers.

FIG. 3 is an autoradiograph using ³²P-labeled probes from 4 clonesobtained from the subtractive hybridization process.

FIG. 4 is flow cytometry profile of spleen adherent cells.

FIGS. 5A and B are Western Blots illustrating the increased expressionof OX-2 antigen after pv immunization. FIG. 5A shows staining with acontrol mouse antibody, anti-mouse CD8a. FIG. 5B shows staining withanti-rat MRC OX-2.

FIG. 6 is a graft showing percent survival versus days post renaltransplantation.

FIG. 7 and SEQ.ID.NO.:20, SEQ.ID.NO.:22 and SEQ.ID.NO.:18 shows the cDNAsequence of rat, mouse and human MRC OX-2, respectively.

FIG. 8 and SEQ.ID.NO.:21, SEQ.ID.NO.:2, and SEQ.ID.NO.:19 shows thededuced protein sequence of rat, mouse and human MRC OX-2 protein,respectively.

FIGS. 9A and 9B are bar graphs showing cytokine production and cellproliferation following stimulation by allogeneic DC using hepatic NPMC.

FIGS. 10A, 10B and 10C are bar graphs showing inhibition of cellproliferation and cytokine production by hepatic NPMC.

FIG. 11 is a bar graph analysis of FACS data showing OX-2 expression ina subpopulation of NPC.

FIG. 12 shows PCR analysis mRNA expression of B7-1, B7-2 and OX-2 invarious hepatic NPMC cell fractions.

FIGS. 13A and 13B are bar graphs showing proliferation and cytokineproduction by NPMC from Flt3L treated mice.

FIG. 14 is a bar graph showing cytokines produced from C3H mice withC57BL16 renal allografts and NPC from Flt3 treated C57BL16 donors.

FIG. 15 is a graph showing inhibition of graft rejection with NPC fromFlt3 treated mice.

FIG. 16 is a graph showing that anti-OX-2 reverses inhibition by NPC.The effect of anti-B7-1, anti-B7-2 and anti-OX-2 on primaryallostimulation is shown.

FIG. 17 is a graph showing that anti-OX-2 mAb reverses inhibition by NPCand inhibits the development of immunoregulatory cells.

FIG. 18A is a photograph showing in situ hybridization with antisenseOX-2 in a 8-11 day placenta from a mouse that has undergone fetal loss.

FIG. 18B is a photograph showing in situ hybridization with antisenseOX-2 in a 8-11 day placenta from a mouse that is not susceptible tospontaneous fetal loss.

DETAILED DESCRIPTION OF THE INVENTION

The present inventor has identified genes that show an increase inexpression following portal venous immunization. These genes play a rolein the development of immune suppression or tolerance and may be usefulin developing therapies for the prevention and treatment of transplantrejection, fetal loss, autoimmune disease or allergies.

Using suppression subtractive hybridization (SSH), the inventor hasisolated a clone that is preferentially expressed in mice receivingallogenic renal grafts along with pre-transplant donor-specificimmunization and that encodes the protein OX-2. The OX-2 protein (alsoknown as MRC OX-2) in rat was described as a 41Kd-47 Kd glycoproteinwhich is expressed on the cell surface of thymocytes, folliculardendritic cells and endothelium, B cells and neuronal cells. Differencesin apparent size of the molecule in different tissues is probably afunction of differential glycosylation. The function of the molecule waspreviously unknown, but DNA and amino acid sequence analysis shows ithas a high degree of homology to molecules of the immunoglobulin genefamily, which includes molecules important in lymphocyte antigenrecognition and cell-cell interaction (e.g. CD4, CD8, ICAMs, VCAMs), aswell as adhesion receptor molecules (NCAMs) in the nervous system.Members of the immunoglobulin superfamily are distinct from othermolecules of the integrin and selectin families, which, at least withinthe immune system, also seem to play critical role in cell recognition,migration and even development of the lymphocyte recognition repertoire(by regulating intra-thymic selection events). It has becomeincreasingly evident that molecules of these different families play animportant role in human disease.

The inventor has shown that administering antibodies to OX-2 inhibitedthe graft survival generally seen following pre-transplant pvimmunization. The inventor has also shown that there is negativeassociation between levels of OX-2 and risk of fetal loss. The inventorhas further shown that OX-2 inhibits cytotoxic cells and IL-2 productionand induces IL-4 production. The data supports the role of OX-2 inimmune suppression.

Therapeutic Methods Inducing Immune Suppression

In one aspect, the present invention provides a method of suppressing animmune response comprising administering an effective amount of an OX-2protein or a nucleic acid sequence encoding an OX-2 protein to an animalin need of such treatment. The invention includes a use of an effectiveamount of an OX-2 protein or a nucleic acid sequence encoding an OX-2protein to suppress an immune response.

The term “OX-2 protein” includes the full length OX-2 protein as well asfragments or portions of the protein. Preferred fragments or portions ofthe protein are those that are sufficient to suppress an immuneresponse. The OX-2 protein also includes fragments that can be used toprepare antibodies.

In a preferred embodiment, the OX-2 protein is prepared as a solublefusion protein. The fusion protein may contain the extracellular domainof OX-2 linked to an immunoglobulin (Ig) Fc Region. The OX-2 fusion maybe prepared using techniques known in the art. Generally, a DNA sequenceencoding the extracellular domain of OX-2 is linked to a DNA sequenceencoding the Fc of the Ig and expressed in an appropriate expressionsystem where the OX-2—FcIg fusion protein is produced. The OX-2 proteinmay be obtained from known sources or prepared using recombinant DNAtechniques. The protein may have any of the known published sequencesfor OX-2. (The sequences can be obtained from GenBank. The humansequence accession no. M17226 X0523; the rat sequence accession no.X01785; and the mouse sequence accession no. AF029214.) The protein mayalso be modified to contain amino acid substitutions, insertions and/ordeletions that do not alter the immunosuppressive properties of theprotein. Conserved amino acid substitutions involve replacing one ormore amino acids of the OX-2 amino acid sequence with amino acids ofsimilar charge, size, and/or hydrophobicity characteristics. When onlyconserved substitutions are made the resulting analog should befunctionally equivalent to the OX-2 protein. Non-conserved substitutionsinvolve replacing one or more amino acids of the OX-2 amino acidsequence with one or more amino acids which possess dissimilar charge,size, and/or hydrophobicity characteristics.

The OX-2 protein may be modified to make it more therapeuticallyeffective or suitable. For example, the OX-2 protein may be cyclized ascyclization allows a peptide to assume a more favourable conformation.Cyclization of the OX-2 peptides may be achieved using techniques knownin the art. In particular, disulphide bonds may be formed between twoappropriately spaced components having free sulfhydryl groups. The bondsmay be formed between side chains of amino acids, non-amino acidcomponents or a combination of the two. In addition, the OX-2 protein orpeptides of the present invention may be converted into pharmaceuticalsalts by reacting with inorganic acids including hydrochloric acid,sulphuric acid, hydrobromic acid, phosphoric acid, etc., or organicacids including formic acid, acetic acid, propionic acid, glycolic acid,lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid,tartaric acid, citric acid, benzoic acid, salicylic acid,benzenesulphonic acid, and tolunesulphonic acids.

Administration of an “effective amount” of the OX-2 protein and nucleicacid of the present invention is defined as an amount effective, atdosages and for periods of time necessary to achieve the desired result.The effective amount of the OX-2 protein or nucleic acid of theinvention may vary according to factors such as the disease state, age,sex, and weight of the animal. Dosage regima may be adjusted to providethe optimum therapeutic response. For example, several divided doses maybe administered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

The term “animal” as used herein includes all members of the animalkingdom including humans.

In one embodiment, the present invention provides a method of inducingimmune tolerance to a transplanted organ or tissue in a recipient animalcomprising administering an effective amount of an OX-2 protein or anucleic acid sequence encoding an OX-2 protein to the recipient animalprior to the transplantation of the organ or tissue. The inventionincludes a use of an effective amount of an OX-2 protein or a nucleicadd sequence encoding an OX-2 protein to induce immune tolerance to atransplanted organ or tissue.

The term “inducing immune tolerance” means rendering the immune systemunresponsive to a particular antigen without inducing a prolongedgeneralized immune deficiency. The term “antigen” means a substance thatis capable of inducing an immune response. In the case of autoimmunedisease, immune tolerance means rendering the immune system unresponsiveto an auto-antigen that the host is recognizing as foreign, thus causingan autoimmune response. In the case of allergy, immune tolerance meansrendering the immune system unresponsive to an allergen that generallycauses an immune response in the host. In the case of transplantation,immune tolerance means rendering the immune system unresponsive to theantigens on the transplant An alloantigen refers to an antigen foundonly in some members of a species, such as blood group antigens. Axenoantigen refers to an antigen that is present in members of onespecies but not members of another. Correspondingly, an allograft is agraft between members of the same species and a xenograft is a graftbetween members of a different species.

The recipient can be any member of the animal kingdom including rodents,pigs, cats, dogs, ruminants, non-human primates and preferably humans.The organ or tissue to be transplanted can be from the same species asthe recipient (allograft) or can be from another species (xenograft).The tissues or organs can be any tissue or organ including heart, liver,kidney, lung, pancreas, pancreatic islets, brain tissue, cornea, bone,intestine, skin and heamatopoietic cells.

The method of the invention may be used to prevent graft versus hostdisease wherein the immune cells in the transplant mount an immuneattack on the recipient's immune system. This can occur when the tissueto be transplanted contains immune cells such as when bone marrow orlymphoid tissue is transplanted when treating leukemias, aplasticanemias and enzyme or immune deficiencies, for example.

Accordingly, in another embodiment, the present invention provides amethod of preventing or inhibiting graft versus host disease in arecipient animal receiving an organ or tissue transplant comprisingadministering an effective amount of an OX-2 protein or a nucleic acidsequence encoding an OX-2 protein to the organ or tissue prior to thetransplantation in the recipient animal. The invention includes a use ofan effective amount of an OX-2 protein or a nucleic acid moleculeencoding an OX-2 protein to prevent or inhibit graft versus hostdisease.

The present inventor has shown that there is an association betweenlevels of OX-2 expression and fertility. In particular the inventor hasshown that low levels (or no levels) of OX-2 is related to fetal loss.Accordingly, the present invention provides a method of preventing orinhibiting fetal loss comprising administering an effective amount of anOX-2 protein or a nucleic acid sequence encoding an OX-2 protein to ananimal in need thereof. The invention includes a use of an effectiveamount of an OX-2 protein on a nucleic acid molecules encoding an OX-2protein to prevent or inhibit fetal loss.

As stated previously, the method of the present invention may also beused to treat or prevent autoimmune disease. In an autoimmune disease,the immune system of the host fails to recognize a particular antigen as“self” and an immune reaction is mounted against the host's tissuesexpressing the antigen. Normally, the immune system is tolerant to itsown host's tissues and autoimmunity can be thought of as a breakdown inthe immune tolerance system.

Accordingly, in a further embodiment, the present invention provides amethod of preventing or treating an autoimmune disease comprisingadministering an effective amount of an OX-2 protein or a nucleic acidsequence encoding an OX-2 protein to an animal having, suspected ofhaving, or susceptible to having an autoimmune disease. The inventionincludes a use of an effective amount of an OX-2 protein on a nucleicacid molecule encoding an OX-2 protein to prevent or inhibit anautoimmune disease.

Autoimmune diseases that may be treated or prevented according to thepresent invention include, but are not limited to, type 1insulin-dependent diabetes mellitus, adult respiratory distresssyndrome, inflammatory bowel disease, dermatitis, meningitis, thromboticthrombocytopenic purpura, Sjögren's syndrome, encephalitis, uveitic,leukocyte adhesion deficiency, rheumatoid arthritis, rheumatic fever,Reiter's syndrome, psoriatic arthritis, progressive systemic sclerosis,primary biniary cirrhosis, pemphigus, pemphigoid, necrotizingvasculitis, myasthenia gravis, multiple sclerosis, lupus erythematosus,polymyositis, sarcoidosis, granulomatosis, vasculitis, perniciousanemia, CNS inflammatory disorder, antigen-antibody complex mediateddiseases, autoimmune haemolytic anemia, Hashimoto's thyroiditis, Gravesdisease, habitual spontaneous abortions, Reynard's syndrome,glomerulonephritis, dermatomyositis, chronic active hepatitis, celiacdisease, autoimmune complications of AIDS, atrophic gastritis,ankylosing spondylitis and Addison's disease.

As stated previously, the method of the present invention may also beused to treat or prevent an allergic reaction. In an allergic reaction,the immune system mounts an attack against a generally harmless,innocuous antigen or allergen. Allergies that may be prevented ortreated using the methods of the invention include, but are not limitedto, hay fever, asthma, atopic eczema as well as allergies to poison oakand ivy, house dust mites, bee pollen, nuts, shellfish, penicillin andnumerous others.

Accordingly, in a further embodiment, the present invention provides amethod of preventing or treating an allergy comprising administering aneffective amount of an OX-2 protein or a nucleic acid sequence encodingan OX-2 protein to an animal having or suspected of having an allergy.The invention includes a use of an effective amount of an OX-2 proteinor a nucleic acid molecule encoding an OX-2 protein to prevent or treatan allergy.

Preventing Immune Suppression

In another aspect, the present invention provides a method of preventingimmune suppression comprising administering an effective amount of anagent that inhibits OX-2 to an animal in need thereof.

There are a large number of situations whereby it is desirable toprevent immune suppression including, but not limited to, the treatmentof infections, cancer and Acquired Immune Deficiency Syndrome.

In one embodiment, the present invention provides a method of preventingimmune suppression comprising administering an effective amount of anagent that binds OX-2 to an animal in need thereof.

In a preferred embodiment, the agent that binds OX-2 is an OX-2 specificantibody. The present inventor has prepared antibodies to OX-2 which aredescribed in Examples 4 and 5. Antibodies to OX-2 may also be preparedusing techniques known in the art such as those described by Kohler andMilstein, Nature 256, 495 (1975) and in U.S. Pat. Nos. RE 32,011;4,902,614; 4,543,439; and 4,411,993, which are incorporated herein byreference. (See also Monoclonal Antibodies, Hybridomas: A New Dimensionin Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol(eds.), 1980, and Antibodies: A Laboratory Manual, Harlow and Lane(eds.), Cold Spring Harbor Laboratory Press, 1988, which are alsoincorporated herein by reference). Within the context of the presentinvention, antibodies are understood to include monoclonal antibodies,polyclonal antibodies, antibody fragments (e.g., Fab, and F(ab′)₂) andrecombinantly produced binding partners.

In another embodiment, the OX-2 inhibitor is an antisenseoligonucleotide that inhibits the expression of OX-2. Antisenseoligonucleotides that are complimentary to a nucleic acid sequence froman OX-2 gene can be used in the methods of the present invention toinhibit OX-2. The present inventor has prepared antisenseoligonucleotides to OX-2 which are described in Example 3.

Consequently, the present invention provides a method of preventingimmune suppression comprising administering an effective amount of anantisense oligonucleotide that is complimentary to a nucleic acidsequence from an OX-2 gene to an animal in need thereof.

The term antisense oligonucleotide as used herein means a nucleotidesequence that is complimentary to its target.

In one embodiment of the invention, the present invention provides anantisense oligonucleotide that is complimentary to a nucleic acidmolecule having a sequence as shown in FIG. 7 and SEQ.ID.NO.:18,SEQ.ID.NO.:20 and SEQ.ID.NO.:22, wherein T can also be U, or a fragmentthereof.

The term “oligonucleotide” refers to an oligomer or polymer ofnucleotide or nucleoside monomers consisting of naturally occurringbases, sugars, and intersugar (backbone) linkages. The term alsoincludes modified or substituted oligomers comprising non-naturallyoccurring monomers or portions thereof, which function similarly. Suchmodified or substituted oligonucleotides may be preferred over naturallyoccurring forms because of properties such as enhanced cellular uptake,or increased stability in the presence of nucleases. The term alsoincludes chimeric oligonucleotides which contain two or more chemicallydistinct regions. For example, chimeric oligonucleotides may contain atleast one region of modified nucleotides that confer beneficialproperties (e.g. increased nuclease resistance, increased uptake intocells), or two or more oligonucleotides of the invention may be joinedto form a chimeric oligonucleotide.

The antisense oligonucleotides of the present invention may beribonucleic or deoxyribonucleic acids and may contain naturallyoccurring bases including adenine, guanine, cytosine, thymidine anduracil. The oligonucleotides may also contain modified bases such asxanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and otheralkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-azacytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine,8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyladenine and other 8-substituted adenines, 8-halo guanines, 8-aminoguanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine andother 8-substituted guanines, other aza and deaza uracils, thymidines,cytosines, adenines, or guanines, 5-trifluoromethyl uracil and5-trifluoro cytosine.

Other antisense oligonucleotides of the invention may contain modifiedphosphorous, oxygen heteroatoms in the phosphate backbone, short chainalkyl or cycloalkyl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages. For example, the antisenseoligonucleotides may contain phosphorothioates, phosphotriesters, methylphosphonates, and phosphorodithioates. In an embodiment of the inventionthere are phosphorothioate bonds links between the four to six3′-terminus bases. In another embodiment phosphorothioate bonds link allthe nucleotides.

The antisense oligonucleotides of the invention may also comprisenucleotide analogs that may be better suited as therapeutic orexperimental reagents. An example of an oligonucleotide analogue is apeptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphatebackbone in the DNA (or RNA), is replaced with a polyamide backbonewhich is similar to that found in peptides (P. E. Nielsen, et al Science1991, 254, 1497). PNA analogues have been shown to be resistant todegradation by enzymes and to have extended lives in vivo and in vitro.PNAs also bind stronger to a complimentary DNA sequence due to the lackof charge repulsion between the PNA strand and the DNA strand. Otheroligonucleotides may contain nucleotides containing polymer backbones,cyclic backbones, or acyclic backbones. For example, the nucleotides mayhave morpholino backbone structures (U.S. Pat. No. 5,034,506).Oligonucleotides may also contain groups such as reporter groups, agroup for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an antisense oligonucleotide. Antisense oligonucleotides may alsohave sugar mimetics.

The antisense nucleic acid molecules may be constructed using chemicalsynthesis and enzymatic ligation reactions using procedures known in theart. The antisense nucleic acid molecules of the invention or a fragmentthereof, may be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed with mRNA or the native gene e.g.phosphorothioate derivatives and acridine substituted nucleotides. Theantisense sequences may be produced biologically using an expressionvector introduced into cells in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense sequences are producedunder the control of a high efficiency regulatory region, the activityof which may be determined by the cell type into which the vector isintroduced.

Compositions

The invention also includes pharmaceutical compositions containing OX-2proteins or nucleic acids for use in immune suppression as well aspharmaceutical compositions containing an OX-2 inhibitor for use inpreventing immune suppression.

Such pharmaceutical compositions can be for intralesional, intravenous,topical, rectal, parenteral, local, inhalant or subcutaneous,intradermal, intramuscular, intrathecal, transperitoneal, oral, andintracerebral use. The composition can be in liquid, solid or semisolidform, for example pills, tablets, creams, gelatin capsules, capsules,suppositories, soft gelatin capsules, gels, membranes, tubelets,solutions or suspensions.

The pharmaceutical compositions of the invention can be intended foradministration to humans or animals. Dosages to be administered dependon individual needs, on the desired effect and on the chosen route ofadministration.

The pharmaceutical compositions can be prepared by per se known methodsfor the preparation of pharmaceutically acceptable compositions whichcan be administered to patients, and such that an effective quantity ofthe active substance is combined in a mixture with a pharmaceuticallyacceptable vehicle. Suitable vehicles are described, for example, inRemington's Pharmaceutical Sciences (Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., USA 1985).

On this basis, the pharmaceutical compositions include, albeit notexclusively, the active compound or substance in association with one ormore pharmaceutically acceptable vehicles or diluents, and contained inbuffered solutions with a suitable pH and iso-osmotic with thephysiological fluids. The pharmaceutical compositions may additionallycontain other agents such as immunosuppressive drugs or antibodies toenhance immune tolerance or immunostimulatory agents to enhance theimmune response.

In one embodiment, the pharmaceutical composition for use in inducingimmune tolerance comprises an effective amount of an OX-2 protein inadmixture with a pharmaceutically acceptable diluent or carrier. TheOX-2 protein is preferably prepared as an immunoadhesion molecule insoluble form which can be administered to the patient. In the case oftissue or organ transplantation, the composition preferably containsOX-2 proteins in soluble form which may be injected intravenously orperfused directly at the site of the transplantation.

In another embodiment, the pharmaceutical composition for use ininducing immune tolerance comprises an effective amount of a nucleicacid molecule encoding an OX-2 protein in admixture with apharmaceutically acceptable diluent or carrier.

The nucleic acid molecules of the invention encoding an OX-2 protein maybe used in gene therapy to induce immune tolerance. Recombinantmolecules comprising a nucleic acid sequence encoding an OX-2 protein,or fragment thereof, may be directly introduced into cells or tissues invivo using delivery vehicles such as retroviral vectors, adenoviralvectors and DNA virus vectors. They may also be introduced into cells invivo using physical techniques such as microinjection andelectroporation or chemical methods such as coprecipitation andincorporation of DNA into liposomes. Recombinant molecules may also bedelivered in the form of an aerosol or by lavage. The nucleic acidmolecules of the invention may also be applied extracellularly such asby direct injection into cells. The nucleic acid molecules encoding OX-2are preferably prepared as a fusion with a nucleic acid moleculeencoding an immunoglobulin (Ig) Fc region. As such, the OX-2 proteinwill be expressed in vivo as a soluble fusion protein.

In another aspect, the pharmaceutical composition for use in preventingimmune suppression comprises an effective amount of an OX-2 inhibitor inadmixture with a pharmaceutically acceptable diluent or carrier. Suchcompositions may be administered as a vaccine either alone or incombination with other active agents or antigens. When used incombination, the OX-2 inhibitors may act like an adjuvant bypotentiating the immune response to the antigen in the vaccine.

In one embodiment, the pharmaceutical composition for use in preventingimmune suppression comprises an effective amount of an antibody to OX-2in admixture with a pharmaceutically acceptable diluent or carrier. Theantibodies may be delivered intravenously.

In another embodiment, the pharmaceutical composition for use inpreventing immune suppression comprises an effective amount of anantisense oligonucleotide nucleic acid complimentary to a nucleic acidsequence from an OX-2 gene in admixture with a pharmaceuticallyacceptable diluent or carrier. The oligonucleotide molecules may beadministered as described above for the compositions containing OX-2nucleic acid sequences.

Murine OX-2

The inventor has cloned and sequenced the murine OX-2 gene. Accordingly,the invention also includes an isolated nucleic acid sequence encoding amurine OX-2 gene and having the sequence shown in FIG. 7 andSEQ.ID.NO.:22.

The term “isolated” refers to a nucleic acid substantially free ofcellular material or culture medium when produced by recombinant DNAtechniques, or chemical precursors, or other chemicals when chemicallysynthesized. The term “nucleic acid” is intended to include DNA and RNAand can be either double stranded or single stranded.

Preferably, the purified and isolated nucleic acid molecule of theinvention comprises (a) a nucleic acid sequence as shown inSEQ.ID.NO.:22, wherein T can also be U; (b) nucleic acid sequencescomplementary to (a); (c) a fragment of (a) or (b) that is at least 15bases, preferably 20 to 30 bases, and which will hybridize to (a) or (b)under stringent hybridization conditions; or (a) a nucleic acid moleculediffering from any of the nucleic acids of (a) or (b) in codon sequencesdue to the degeneracy of the genetic code.

It will be appreciated that the invention includes nucleic acidmolecules encoding truncations of the murine OX-2 proteins of theinvention, and analogs and homologs of the proteins of the invention andtruncations thereof, as described below. It will further be appreciatedthat variant forms of the nucleic acid molecules of the invention whicharise by alternative splicing of an mRNA corresponding to a cDNA of theinvention are encompassed by the invention.

An isolated nucleic acid molecule of the invention which is DNA can alsobe isolated by selectively amplifying a nucleic acid encoding a novelprotein of the invention using the polymerase chain reaction (PCR)methods and cDNA or genomic DNA. It is possible to design syntheticoligonucleotide primers from the nucleic acid molecules as shown in FIG.7 and SEQ.ID.NO.:22 for use in PCR. A nucleic acid can be amplified fromcDNA or genomic DNA using these oligonucleotide primers and standard PCRamplification techniques. The nucleic acid so amplified can be clonedinto an appropriate vector and characterized by DNA sequence analysis.It will be appreciated that cDNA may be prepared from mRNA, by isolatingtotal cellular mRNA by a variety of techniques, for example, by usingthe guanidinium-thiocyanate extraction procedure of Chirgwin et al.,Biochemistry, 18, 5294-5299 (1979). cDNA is then synthesized from themRNA using reverse transcriptase (for example, Moloney MLV reversetranscriptase available from Gibco/BRL, Bethesda, Md., or AMV reversetranscriptase available from Seikagaku America, Inc., St. Petersburg,Fla.).

An isolated nucleic acid molecule of the invention which is RNA can beisolated by cloning a cDNA encoding a novel protein of the inventioninto an appropriate vector which allows for transcription of the cDNA toproduce an RNA molecule which encodes a OX-2 protein of the invention.For example, a cDNA can be cloned downstream of a bacteriophagepromoter, (e.g. a T7 promoter) in a vector, cDNA can be transcribed invitro with T7 polymerase, and the resultant RNA can be isolated bystandard techniques.

A nucleic acid molecule of the invention may also be chemicallysynthesized using standard techniques. Various methods of chemicallysynthesizing polydeoxynucleotides are known, including solid-phasesynthesis which, like peptide synthesis, has been fully automated incommercially available DNA synthesizers (See e.g., Itakura et al. U.S.Pat. No. 4,598,049; Caruthers et al. U.S. Pat. No. 4,458,066; andItakura U.S. Pat. Nos. 4,401,796 and 4,373,071).

The sequence of a nucleic acid molecule of the invention may be invertedrelative to its normal presentation for transcription to produce anantisense nucleic acid molecule. Preferably, an antisense sequence isconstructed by inverting a region preceding the initiation codon or anunconserved region. In particular, the nucleic acid sequences containedin the nucleic acid molecules of the invention or a fragment thereof,preferably a nucleic acid sequence shown in FIG. 7 and SEQ.ID.NO.:22 maybe inverted relative to its normal presentation for transcription toproduce antisense nucleic acid molecules.

The antisense nucleic acid molecules of the invention or a fragmentthereof, may be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed with mRNA or the native gene e.g.phosphorothioate derivatives and acridine substituted nucleotides. Theantisense sequences may be produced biologically using an expressionvector introduced into cells in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense sequences are producedunder the control of a high efficiency regulatory region, the activityof which may be determined by the cell type into which the vector isintroduced.

The invention also provides nucleic acids encoding fusion proteinscomprising an OX-2 protein of the invention and a selected protein, or aselectable marker protein.

The invention further includes an isolated protein which has the aminoacid sequence as shown in FIG. 8 and SEQ.ID.NO.:2.

Within the context of the present invention, a protein of the inventionmay include various structural forms of the primary protein which retainbiological activity. For example, a protein of the invention may be inthe form of acidic or basic salts or in neutral form. In addition,individual amino acid residues may be modified by oxidation orreduction.

In addition to the full length amino acid sequence (FIG. 8 andSEQ.ID.NO.:2), the protein of the present invention may also includetruncations of the protein, and analogs, and homologs of the protein andtruncations thereof as described herein. Truncated proteins may comprisepeptides of at least fifteen amino acid residues.

Analogs of the protein having the amino acid sequence shown in FIG. 8and SEQ.ID.NO.:2, and/or truncations thereof as described herein, mayinclude, but are not limited to an amino acid sequence containing one ormore amino acid substitutions, insertions, and/or deletions. Amino acidsubstitutions may be of a conserved or non-conserved nature. Conservedamino acid substitutions involve replacing one or more amino acids ofthe proteins of the invention with amino acids of similar charge, size,and/or hydrophobicity characteristics. When only conserved substitutionsare made the resulting analog should be functionally equivalent.Non-conserved substitutions involve replacing one or more amino acids ofthe amino acid sequence with one or more amino acids which possessdissimilar charge, size, and/or hydrophobicity characteristics.

One or more amino acid insertions may be introduced into the amino acidsequences shown in FIG. 8 and SEQ.ID.NO.:2. Amino acid insertions mayconsist of single amino acid residues or sequential amino acids rangingfrom 2 to 15 amino acids in length. For example, amino acid insertionsmay be used to render the protein is no longer active. This proceduremay be used in vivo to inhibit the activity of a protein of theinvention.

Deletions may consist of the removal of one or more amino acids, ordiscrete portions from the amino acid sequence shown in FIG. 8. Thedeleted amino acids may or may not be contiguous. The lower limit lengthof the resulting analog with a deletion mutation is about 10 aminoacids, preferably 100 amino acids.

Analogs of a protein of the invention may be prepared by introducingmutations in the nucleotide sequence encoding the protein. Mutations innucleotide sequences constructed for expression of analogs of a proteinof the invention must preserve the reading frame of the codingsequences. Furthermore, the mutations will preferably not createcomplementary regions that could hybridize to produce secondary mRNAstructures, such as loops or hairpins, which could adversely affecttranslation of the receptor mRNA.

Mutations may be introduced at particular loci by synthesizingoligonucleotides containing a mutant sequence, flanked by restrictionsites enabling ligation to fragments of the native sequence. Followingligation, the resulting reconstructed sequence encodes an analog havingthe desired amino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site specific mutagenesisprocedures may be employed to provide an altered gene having particularcodons altered according to the substitution, deletion, or insertionrequired. Deletion or truncation of a protein of the invention may alsobe constructed by utilizing convenient restriction endonuclease sitesadjacent to the desired deletion. Subsequent to restriction, overhangsmay be filled in, and the DNA religated. Exemplary methods of making thealterations set forth above are disclosed by Sambrook et al (MolecularCloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor LaboratoryPress, 1989).

The invention also contemplates isoforms of the proteins of theinvention. An isoform contains the same number and kinds of amino acidsas a protein of the invention, but the isoform has a different molecularstructure. The isoforms contemplated by the present invention are thosehaving the same properties as a protein of the invention as describedherein.

The present invention also includes a protein of the inventionconjugated with a selected protein, or a selectable marker protein toproduce fusion proteins. Additionally, immunogenic portions of a proteinof the invention are within the scope of the invention.

The proteins of the invention (including truncations, analogs, etc.) maybe prepared using recombinant DNA methods. Accordingly, the nucleic acidmolecules of the present invention having a sequence which encodes aprotein of the invention may be incorporated in a known manner into anappropriate expression vector which ensures good expression of theprotein. Possible expression vectors include but are not limited tocosmids, plasmids, or modified viruses (e.g. replication defectiveretroviruses, adenoviruses and adeno-associated viruses), so long as thevector is compatible with the host cell used. The expression vectors are“suitable for transformation of a host cell”, means that the expressionvectors contain a nucleic acid molecule of the invention and regulatorysequences selected on the basis of the host cells to be used forexpression, which is operatively linked to the nucleic acid molecule.Operatively linked is intended to mean that the nucleic acid is linkedto regulatory sequences in a manner which allows expression of thenucleic acid.

The invention therefore contemplates a recombinant expression vector ofthe invention containing a nucleic acid molecule of the invention, or afragment thereof, and the necessary regulatory sequences for thetranscription and translation of the inserted protein-sequence. Suchexpression vectors may be useful in the above-described therapies usinga nucleic acid sequence encoding an OX-2 protein. Suitable regulatorysequences may be derived from a variety of sources, including bacterial,fungal, or viral genes (For example, see the regulatory sequencesdescribed in Goeddel, Gene Expression Technology: Methods in Enzymology185, Academic Press, San Diego, Calif. (1990). Selection of appropriateregulatory sequences is dependent on the host cell chosen, and may bereadily accomplished by one of ordinary skill in the art. Examples ofsuch regulatory sequences include: a transcriptional promoter andenhancer or RNA polymerase binding sequence, a ribosomal bindingsequence, including a translation initiation signal. Additionally,depending on the host cell chosen and the vector employed, othersequences, such as an origin of replication, additional DNA restrictionsites, enhancers, and sequences conferring inducibility of transcriptionmay be incorporated into the expression vector. It will also beappreciated that the necessary regulatory sequences may be supplied bythe native protein and/or its flanking regions.

The invention further provides a recombinant expression vectorcomprising a DNA nucleic acid molecule of the invention cloned into theexpression vector in an antisense orientation. That is, the DNA moleculeis operatively linked to a regulatory sequence in a manner which allowsfor expression, by transcription of the DNA molecule, of an RNA moleculewhich is antisense to a nucleotide sequence comprising the nucleotidesas shown in FIG. 7 and SEQ.ID.NO.:22. Regulatory sequences operativelylinked to the antisense nucleic acid can be chosen which direct thecontinuous expression of the antisense RNA molecule.

The recombinant expression vectors of the invention may also contain aselectable marker gene which facilitates the selection of host cellstransformed or transfected with a recombinant molecule of the invention.Examples of selectable marker genes are genes encoding a protein such asG418 and hygromycin which confer resistance to certain drugs,β-galactosidase, chloramphenicol acetyltransferase, or fireflyluciferase. Transcription of the selectable marker gene is monitored bychanges in the concentration of the selectable marker protein such asβ-galactosidase, chloramphenicol acetyltransferase, or fireflyluciferase. If the selectable marker gene encodes a protein conferringantibiotic resistance such as neomycin resistance transformant cells canbe selected with G418. Cells that have incorporated the selectablemarker gene will survive, while the other cells die. This makes itpossible to visualize and assay for expression of recombinant expressionvectors of the invention and in particular to determine the effect of amutation on expression and phenotype. It will be appreciated thatselectable markers can be introduced on a separate vector from thenucleic acid of interest.

The recombinant expression vectors may also contain genes which encode afusion moiety which provides increased expression of the recombinantprotein; increased solubility of the recombinant protein; and aid in thepurification of a target recombinant protein by acting as a ligand inaffinity purification. For example, a proteolytic cleavage site may beadded to the target recombinant protein to allow separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein.

Recombinant expression vectors can be introduced into host cells toproduce a transformant host cell. The term “transformant host cell” isintended to include prokaryotic and eukaryotic cells which have beentransformed or transfected with a recombinant expression vector of theinvention. The terms “transformed with”, “transfected with”,“transformation” and “transfection” are intended to encompassintroduction of nucleic acid (e.g. a vector) into a cell by one of manypossible techniques known in the art. Prokaryotic cells can betransformed with nucleic acid by, for example, electroporation orcalcium-chloride mediated transformation. Nucleic acid can be introducedinto mammalian cells via conventional techniques such as calciumphosphate or calcium chloride coprecipitation, DEAE-dextran-mediatedtransfection, lipofectin, electroporation or microinjection. Suitablemethods for transforming and transfecting host cells can be found inSambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition,Cold Spring Harbor Laboratory press (1989)), and other laboratorytextbooks.

Suitable host cells include a wide variety of prokaryotic and eukaryotichost cells. For example, the proteins of the invention may be expressedin bacterial cells such as E. coli, insect cells (using baculovirus),yeast cells or mammalian cells. Other suitable host cells can be foundin Goeddel, Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1991).

The proteins of the invention may also be prepared by chemical synthesisusing techniques well known in the chemistry of proteins such as solidphase synthesis (Merrifield, 1964, J. Am. Chem. Assoc. 85:2149-2154) orsynthesis in homogenous solution (Houbenweyl, 1987, Methods of OrganicChemistry, ed. E. Wansch, Vol. 15 I and II, Thieme, Stuttgart).

The following non-limiting examples are illustrative of the presentinvention:

EXAMPLES Example 1

This example demonstrates the increased expression of certain genesfollowing pv immunization.

Mice:

C3H/HEJ and C57BL/6 mice were purchased from The Jackson Laboratory, BarHarbor, Me. Mice were housed five/cage and allowed food and water adlibitum. All mice were used at 8-12 weeks of age.

Monoclonal Antibodies:

The following monoclonal antibodies (Mabs) from Pharmingen (San Diego,Calif.) were used: anti-IL-2 (JES6-1A12; biotinylated JES6-5H4);anti-IL-4 (11B11; biotinylated BVD6-24G2); anti-IFNγ (R4-6A2;biotinylated XMG1.2); anti-IL-10 (JES5-2A5; biotinylated SXC-1,Pharmingen, San Diego, Calif.); mouse IgG1 isotype control (clone 107.3,BALB/c anti-TNP). Strepavidin horse radish peroxidase and recombinantmouse GM-CSF was also purchased from Pharmingen (San Diego, Calif.).

NLDC-145 (anti-mouse dendritic cells), and F(ab′)₂ rabbit anti-rat IgGFITC conjugate (non-cross reactive with mouse IgG), or F(ab′)₂ rabbitanti-mouse IgG PE was obtained from Serotec, Canada.

Rabbit complement, L3T4, anti-thy1.2, anti-Ly2.2, anti-Ly2.1 (mouseIgG3), FITC-MAC-1 and mouse IgG1 anti-rat OX-2 were obtained fromCedarlane Labs, Hornby, Ontario.

Anti-CD28 (PV-1) and anti-CTLA (UC10-4F10-11) were obtained from Drs. C.June and J. Bluestone respectively, while anti-B7-1, anti-B7-2 wereobtained from Dr. G. Powers. High titres of all 4 of the latterantibodies were produced by in vitro culture in a CELLMAX system (CELLCOInc., Germantown, Md., USA).

Preparation of Cells:

Spleen, Peyer's Patch (PP) and mesenteric lymph node (MLN) cellsuspensions were prepared aseptically from individual mice of thedifferent treated groups in each experiment.

Where dendritic cells were obtained by culture of bone marrow cells invitro the following technique was used (Gorczynski et al., 1996a). Bonemarrow plugs were aspirated from the femurs of donor male C57BL/6 (orBALB/c) mice, washed and resuspended in αF10. Cells were treatedsequentially with a mixture of antibodies (L3T4, anti-thy1.2,anti-Ly2.2) and rabbit complement and dead cells removed bycentrifugation over mouse lymphopaque (Cedarlane Labs, Ontario). Cellswere washed×3 in αF10, and cultured in 10ml αF10 in tissue cultureflasks, at a concentration of 2×10⁶/ml with 500U/ml recombinant murineGM-CSF (Pharmingen, USA). Fresh GM-CSF was added at 36 hr intervals.Cells were separated over lymphopaque on days 3.5 and 7 of culture,again reculturing in αF10 with recombinant GM-CSF. At 10 days an aliquotof the sample was stained with NLDC-145 and FITC anti-rat IgG, anti-OX-2and PE anti-mouse IgG, FITC-anti-B7-1 or FITC anti-B7-2. Mean stainingwith these antibodies using cells harvested from such cultures has been93%±7%, 14%±5%, 78%±9% and 27%±6% respectively. Remaining cells werewashed, and injected into the portal vein as described.

Portal Vein Immunizations and Renal Transplantation:

The pv immunizations and renal transplantation were performed asdescribed earlier (Gorczynski et al., 1994). All C3H mice received pv/ivimmunization with 15×10⁶ C57BL/6 10-day cultured, bone marrow derived,dendritic cells, followed by C57BL/6 kidney transplantation. Animalsreceived 1 intramoscular (im) injection with 10 mg/Kg cyclosporin A onthe day of transplantation. Mice were sacrificed for tissue harvest andRNA preparation 5 days after transplantation. In other studies animalswere sacrificed as described in the text.

Where monoclonal antibodies were injected into transplanted mice,animals received 100 mg intravenous (iv) at 2 day intervals (×4injections) beginning within 2 hours of transplantation.

Cytokine Production From Spleen Cells of Transplanted Mice:

In cultures used to assess induction of cytokine production spleenresponder cells stimulated with irradiated (2000R) C57BL/6 spleenstimulator cells in triplicate in αF10 have been used. In multiplestudies significant quantitative or qualitative differences in cytokineproduction from spleen, lymph node or Peyer's Patch of transplanted micehave not been seen. (Gorczynski et al., 1994b). Supernatants were pooledat 40 hr from replicate wells and assayed in triplicate in ELISA assaysfor lymphokine production. AU capture antibodies, biotinylated detectionantibodies, and recombinant cytokines were obtained from Pharmingen (SanDiego, Calif.—see above).

For IFNγ the assay used flat-bottomed 96-well Nunc plates (Gibco, BRL)coated with 100 ng/ml R4-6A2. Varying volumes of supernatant were boundin triplicate at 4° C., washed×3, and biotinylated anti-IFNγ (XMG1.2)added. After washing, plates were incubated with strepavidin-horseradish peroxidase (Cedarlane Labs), developed with appropriate substrateand OD₄₀₅ determined using an ELISA plate reader. IL-10 was assayedusing a similar ELISA system with JES5-2A5 as the capture antibody, andbiotinylated SXC-1 as developing antibody. Each assay reliably detectedcytokine levels in the range 0.01 to 0.1 ng/ml. ELISA assays for IL-2and IL-4 used JES6-1A12 and 11B11 as capture antibodies, withbiotinylated JES6-5H4 or BVD6-24G2 as developing antibodies. Sensitivityof detection was 10 pg/ml for each cytokine.

Oligonucleotide Primers

The primers used for PCR amplification for β-actin, and differentcytokines, are described in previous publications (Gorczynski, R. M.,1995a; Gorczynski, R. M., 1995b; Gorczynski, R. M., 1996a). In addition,the following oligonucleotides were synthesized.

cDNA synthesis primer for driver ds cDNA (DP): 5′-TTTTGTACAAGCTT₃₀-3′(SEQ.ID.NO.:3) Adapter 1 (Ad1):5′-CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT-3′ (SEQ.ID.NO.:4)Adapter 2 (Ad2): 5′-TGTAGCGTGAAGACGACAGAAAGGGCGTGGTGCGGAGGGCGGT-3′(SEQ.ID.NO.:5) PCR Primer1 (P1): 5′-CTAATACGACTCACTATAGGGC-3′(SEQ.ID.NO.:6) Nested Primer 1 (NP1): 5′-TCGAGCGGCCGCCCGGGCAGGT-3′(SEQ.ID.NO.:7) PCR Primer2 (P2): 5′-TGTAGCGTGAAGACGACAGAA-3′(SEQ.ID.NO.:8) Nested Primer 2 (NP2): 5′-AGGGCGTGGTGCGGAGGGCGGT-3′(SEQ.ID.NO.:9)

Driver and Tester Preparation:

RNA was extracted from pooled mesenteric lymph node (MLN) and Peyer'sPatches (PP) of 5/group renal transplant mice with iv or pvimmunization. Poly(A)⁺mRNA was prepared from the driver (iv) group, and2 mg material used for ds cDNA synthesis with 1 ng DP primer and a cDNASynthesis Kit (Clontech) with T4 DNA polymerase. The final cDNApreparation was digested with RsaI in a 50 ml reaction mixture with 15units enzyme (GIBCO) for 3 hrs, and the cDNA phenol-extracted, ethanolprecipitated, and resuspended in 7 ml of deionized water (concentrationapproximately 300 ng/ml).

RsaI digested ds tester cDNA (pv group) was prepared in a similarfashion. 50 ng of tester cDNA diluted in TE buffer was ligated with 2 mlof Ad1 and Ad2 (each at 10 mM) in separate ligation reactions at 16° C.for 18 hrs with 50 Units/ml T4 ligase. Thereafter 1 ml of 0.2M EDTA wasadded, the mixture heated at 70° C. for 5 min to inactivate the ligase,and the product stored at −70° C.

Subtractive Hybridization and PCR Amplification:

600 ng driver (iv) ds cDNA was added to each of two tubes containing 20ng Ad1- and Adi2-ligated pv cDNA. The samples were mixed, precipitatedwith ethanol, resuspended in hybridization buffer, overlaid with mineraloil and denatured/annealed in standard fashion. The two independentsamples were then combined, 200 ng fresh driver cDNA added to allow forfurther enrichment of differentially expressed mRNAs, and the mixtureagain denatured and annealed for 10 hrs at 68° C. The final sample wasdiluted in Hepes buffer with EDTA and stored at −20° C.

After subtraction two PCR amplifications were performed on thesubtracted cDNA. In the first 1 ml of subtracted cDNA was amplifiedusing 1 ml each of P1 and P2. The conditions for amplification were asdescribed by Diatchenko. The amplified products were diluted 10-fold indeionized water and 1 ml of product used for further amplification usingthe nested primers (NP1 and NP2) and a 10 cycle amplification reaction.Aliquots of; the original driver/tester and subtracted cDNAs were usedfor PCR reactions with control oligonucleotide primers (β-actin) forknown “housekeeping genes”, and with primers for genes whose expressionhas been previously documented to be different in iv/pv immunized mice.These data are shown in FIGS. 1 and 2.

FIG. 1 shows PCR validation of suppressive subtractive hybridization.Samples from unsubtracted (lanes 1, 3, 5 and 7) or subtracted (lanes 2,4, 6 and 8) mRNA were reverse transcribed and tested in PCR with b-actinprimers for different PCR cycle times. Lanes 1 and 2: 15 cycles; lanes 3and 4: 20 cycles; lanes 5 and 6: 25 cycles; lanes 7 and 8: 30 cycles.

FIG. 2 shows PCR validation of suppressive subtractive hybridization.Samples from unsubtracted (lanes 2 and 4) or subtracted (lanes 3 and 5)mRNA were tested as in FIG. 1, except primers used were for IL-10, anddifferent cycle times are shown. Lanes 2 and 3: 20 cycles; lanes 4 and5: 30 cycles, lane 1: mol. wt. standard.

In addition, cloning of the subtracted cDNA was performed as follows.

Cloning and Further Analysis of Subtracted cDNA:

The PCR amplified cDNA was cloned with a TA cloning kit (Invitrogen,California) by directly ligating into the PCR II vector. Ligation wasperformed at an insert:vector ratio of 3:1 in 1×ligation buffer with T4ligase (3 U/μl) overnight at 14° C. Ligation products were then insertedinto INFαF′ competent Escherichia Coli using a standard transformationprotocol, and selected with ampicillin on plates containing X-gal(5-bromo-4-chloro-3-indolyl-D-galactoside). Miniprep plasmid DNA waspurified with a Plasmid extraction Spin kit (Qiagen, Germany) and cutwith EcoR I restriction enzyme to determine whether the plasmidscontained the expected insert. Plasmids with inserts were sequenced bythe dideoxy sequencing method using a T7 sequencing kit (PharmaciaBiotech, Canada). Nucleic acid homology searches were performed usingthe BLAST program at the National Center for Biotechnology Information(NIH, Bethesda, USA).

Further analyses of cloned material, using Northern hybridization, wasas follows. Inserts in pCRII were amplified for 12 cycles using nestedPCR primers. The amplified material was purified using Qiaquick Spin PCRPurification Kits (Qiagen), ³²P-labeled by random priming, and used as aprobe for Northern hybridization with 20 mg samples of the original (andfresh) iv or pv total RNA. Hybridization was performed in 5 ml ofExpressHyb solution (Clontech) with a minimum of 5×10⁶ cpm per 100 ngcDNA probe and 0.1 mg/ml sonicated heat-denatured salmon sperm DNA.Filters were washed 4 times, each at 15 min at 27° C. with 1×SSC and0.1% SDS, followed by a high stringency wash at 42° C. for 30 min with0.2×SSC and 0.1% SDS. Exposure times varied from 18 hrs to 6 days. FIG.3 shows an autoradiograph using ^(32g)P-labeled probes prepared from 4clones obtained using the subtraction hybridization approach describedabove (with pv cDNA as tester material and iv cDNA as driver). A labeledcontrol probe was prepared with a PCR amplicon for mouse b-actin. TotalRNA was prepared from mice receiving iv or pv immunization andequivalent amounts loaded in replicate lanes as shown, with gelsdeveloped from 18 hours (clone #28) to 6 days (#71). Clone 8 is mosthomologous with mouse poly (A) binding protein. Clone 16 is mosthomologous with rat MRC OX-2. Clone 28 is most homologous with humanzinc-finger protein. Clone 71 has no homologous sequence.

Western Blotting Protocol:

The technique used was essentially that described by Sandhu et al.(1991) as modified by Bronstein et al. (1992). Samples were obtained 14days post renal transplantation, using groups described in FIG. 5. Freshrat thymus cells were used as control. Samples were electrophoresed in12% SDS-PAGE and transferred to PVDF membranes (Novex Co., San Diego,Calif.) prior to addition of primary antibody. A commercial anti-ratOX-2 was used as test reagent; control antibody was an antibody to mouseCD8a. The developing antibody used was a commercial horse-radishperoxidase labeled anti-mouse IgG. All reagents were obtained fromCedarlane Labs (Hornby, Ontario, Canada).

DNA Sequence Homology Comparison:

Comparison of mouse OX-2 with known cDNA sequences for B7-1, B7-2, CD28and CTLA-4 was performed using a DNASIS program (version 2.0).

Results

Evaluation of Suppression Subtraction Hybridization(SSH) Technique

In order to evaluate the efficacy of the SSH technique used, theinventor used his previous evidence that, by PCR analysis, increasedexpression of mRNA for IL-10 genes was evident in lymphoid tissue frompv immunized mice. Accordingly, a dilution analysis of cDNA from thetester, driver and subtracted material, using PCR primers for β-actinand IL-10 was performed. As shown in FIG. 1, after SSH there was adetectable signal for β-actin in subtracted material only after 35cycles of amplification. By contrast, a signal was present in theunsubtracted material after only 15 cycles. Using additionalquantitative measures of template, it was found to correspond to some1000-10,000 depletion of β-actin mRNA. In a separate study, analyzingIL-10 mRNA (FIG. 2), significant enrichment of IL-10 mRNA was found asdetermined by comparison of the amplification detected at 30 cycles insubtracted/unsubtracted material (see lanes 4 and 5, FIG. 2).

In a further test of the efficiency of subtraction the mixture ofunsubtracted and subtracted tester (pv) cDNA was labeled and hybridizedto Northern blots of iv (tester) and pv (driver) total RNA. The results(data not shown) indicated that the subtracted tester cDNA probe didindeed produce a significantly stronger signal with the tester RNA.Given the evidence that for any cDNA species to produce a signal in aNorthern blot it must represent a concentration greater than 0.1-0.3% ofthe cDNA mixture, these data are again consistent with our havingproduced a high level of enrichment of pv-specific cDNA, with aconcomitant reduction in abundant cDNAs common between tester (pv) anddriver (iv) material.

Detection of Unique cDNA Fragments in Tissue From pv Immunized Mice

The efficiency and validity of SSH for detection of cDNAs unique to thetissue sample from the pv immunized mice was further confirmed aftercloning and sequence analysis of selected tester-specific cDNAs. 10randomly selected cDNA clones (of 66 sequenced) were used to probemultiple preparations of pv or iv whole RNA. All revealed unique mRNAsexpressed preferentially in the pv samples. Autoradiograms from 4 ofthese Northern blots, along with a β-actin probe as control, are shownin FIG. 3. Exposure times from 18 hrs to 6 days were used which wereinterpreted as indicative of pv specific cDNAs of different abundance inthe samples of interest.

The cDNA inserts of the 4 clones shown, along with the other 62 clones,were partially sequenced and analyzed for homology in the GenBank andEMBL data bases. A summary of these data are shown in Table 1. Note thatsome 30 cDNA fragments had at least 50% homology (BLAST score>250 overat least 50 nt) with other described sequences. A further 14 clonesshowed similar homology with known rat/human genes. Both sets mayrepresent members of different gene families. An additional 22 clonesdemonstrated no significant matches with entries in the database, andthus may represent novel genes up-regulated after pv immunization. Thatthe data shown are a minimal estimate of such differentially expressedgenes is evident from the fact that homology with IL-4 or IL-10 genesequences (mRNAs known to be over-expressed following pvimmunization—see also FIG. 2) were NOT detected in any of the 66 clonesanalyzed.

The sequence homology for the clones shown in FIG. 3 (>80% homology overthe compared sequence) led to the further characterization of theseclones. Clone 8 was shown to be most homologous with mouse poly (A)binding protein; done 16 was shown to be most homologous with rat MRCOX-2; and clone 28 was shown to be most homolgous with human zinc-fingerprotein. No homologous sequence was found for clone 71. In the data thatfollows, the analysis of one of these clones which showed homology to arat cDNA (for OX-2, a molecule previously characterized as beingpreferentially expressed on rat thymocytes and dendritic cells) isdescribed. The rationale for further investigation of this clone lies indata showing that infusion of dendritic cells via the portal vein is apotent method for prolonging allograft survival in our model systems.Note, however, that while the bone marrow derived dendritic cells thatwere infused via the portal vein themselves express OX-2 (see above),identical data has been obtained in Northern gels to those shown in FIG.3 using tissue harvested from mice receiving, as the earlieststudies(1-5) irradiated spleen cells (OX-2³¹ by FACS analysis) via theportal vein. In addition, in both situations, OX-2 mRNA was not detectedby this suppression subtraction hybridization approach when we usedtissue harvested at 0.5-2.5 days post transplantation. These results areconsistent with the idea that the OX-2 signal detected is a result ofnovel increased expression in cells following pv immunization.

Probing a cDNA Library From Tissue From pv Immunized Mice for Expressionof the Murine Equivalent of Rat OX-2

A cDNA library was constructed from mRNA prepared from a pool of 5 C3Hmice receiving pv immunization with 25×10⁶ irradiated (2000 Rads)C57BL/6 bone marrow cells followed by renal transplantation as describedin the Materials and Methods, using a kit purchased from ClonTech.Clones were plated in LB medium and probed with the ^(32g)P-labeledamplicon described in FIG. 3 as showing homology with rat OX-2. A 1.3 Kbclone was detected, amplified, and shown after ³²P labeling to detect adifferentially expressed product by Northern gel analysis. Aftersequencing using an automated DNA sequencer and fluorescent-labeleddeoxynucleotides, this 1.3 Kb fragment was found to share->95% homologywith the region encoding the 3′untranslated region of the rat OX-2 mRNAas determined from the GeneBank sequence for rat OX-2.

Using a primer construct program, a 5′PCR primer representing positions1-19 of the rat GeneBank sequence (corresponding to a portion of the5′untranslated region, and the leader sequence) and 3′ primers from ourcharacterized mouse sequence were synthesized, and long-distanceamplification performed to produce an amplicon predicted to encode theopen-reading-frame (ORF) of the murine equivalent of the rat OX-2 gene.This amplicon was determined (as expected) to be of some 1.4 Kb length.Automated sequencing produced a full-length sequence for the mousehomologue of the rat MRC OX-2 gene, including an ORF with >90% homology(predicted amino acid sequence) with the corresponding rat product,along with the 3′untranslated region. This sequence has been submittedto the Genebank (accession number AF004023).

Using a DNASIS program the predicted mouse protein sequence has some 51%homology with B7-1 and B7-2, 48% with CD28 and 54% with CTLA4(unpublished).

Evidence for an Important Role for the Expressed OX-2 Homologue inProlonged Graft Survival Following pv Immunization

In an attempt to define the potential importance of the product encodedby the OX-2 gene we used a commercial antibody to rat OX-2 in atransplant model in mice receiving pv immunization and renaltransplantation. In the first such study, it was asked whether there wasevidence for specifically increased expression of the OX-2 moleculefollowing pv immunization. By FACS analysis, using dual staining ofhepatic mononuclear cells and spleen cells with OX-2 and NLDC145,similar numbers of NLDC145⁺ cells in liver or spleen samples from iv andpv immunized mice were found, (5×10⁵ and 6.5×10⁶ respectively), but a4-fold increase in the numbers of OX-2⁺ NLDC145⁺ following pvimmunization. FIG. 4 shows a flow cytometry profile of spleen adherentcells from iv immunized/grafted mice (panels A and B) or pvimmunized/grafted mice (panels C and D). Cells were harvested 7 daysafter transplantation and stained with NLDC145 and F(ab′)₂FITC-anti-ratIgG, as well as with control (done 107.3) mouse IgG1 serum (left handpanels) or anti-OX-2 (right hand panels) and F(ab′)₂PE-anti-mouse IgG.Data are representative of one of three different studies. Values shownrepresent the total cell population in each quadrant. The absolutenumbers (×10⁵) of double positive cells in the liver or spleen of pvimmunized mice were 3.2±0.5 and 39±8 respectively (see FIG. 4 for FACSprofiles of spleen adherent cells). This 4-fold increase was seenregardless of the cells used for pv immunization, either bone marrowderived dendritic cells (some 20% OX-2⁺—see above) or irradiated wholespleen lymphoid cells (OX-2⁺), suggesting that they were not merelydetecting surviving OX-2⁻ (donor) cells, but novel expression of OX-2 invivo.

Western blot, FIG. 5, shows increased expression of OX-2 antigen afterpv immunization. The technique used for Western blotting is previouslydescribed. Samples were obtained 14 days post renal transplantation,using the groups described in FIG. 6. Fresh rat thymus cells (lane 5)were used as control. Lanes 1 and 2 represent samples pooled from 3donors/group (iv immunized; pv immunized +infusion of anti-OX-2respectively). Samples in lanes 3 and 4 are from individual micereceiving pv immunization and renal transplantation only (no antibodytreatment). Staining with anti-rat MRC OX-2 is shown in FIG. 5B; with acontrol antibody (to mouse Ly2.1), anti-mouse cD8a, shown in FIG. 5A.The developing antibody used was a commercial horse-radish peroxidaselabeled anti-mouse IgG. No signal was seen using the mouse IgG1 isotypecontrol clone 107.3 (BALB/c anti-TNP)—data not shown. Data arerepresentative of 1 of 3 equivalent studies.

Western blotting (see FIGS. 5A and 5B) of samples prepared from thespleen of iv vs pv immunized and grafted mice 14 days following renaltransplantation revealed staining of a band migrating with estimatedmolecular weight 43 Kd, in agreement with data elsewhere reportingextensive glycosylation of this molecule in isolates from rat thymus. Inmice receiving pv immunization along with in vivo treatment withanti-OX-2, no detectable signal was seen in Western blots (see lane 2,FIG. 5). No staining was seen with a murine IgG1 isotype control (BALB/canti-TNP, done 107.3: unpublished), making it unlikely that the bandobserved was Fc receptor.

FIG. 6 is a graft showing percent survival versus days post renaltransplantation. Commercial anti-OX-2 monoclonal antibody, but notanti-mouse CD28 or anti-mouse CTLA4, reverses the graft prolongationfollowing donor-specific pv immunization. Groups of 6 C3H mice receivedC57BL/6 renal allografts with no other treatment (cyclosporin A only,-⋄-), or additional pv immunization with 15×10⁶ C57BL/6 bone marrowderived dendritic cells (-□-)as described previously. Subsets of theselatter mice received iv injection (every second day ×4 injections) with100 mg/mouse of a commercial anti-rat OX-2 monoclonal antibody (-S-) orthe isotype control (clone 107.3, -∇-), or of antibodies to mouse CD28(--) or CTLA4 (-*-). The animal survival for the different groups shownare pooled from 2 studies. Note that the mouse isotype control itselfproduced no modification of the increased renal graft survival followingpv immunization. *p<0.02, Mann-Whitney U-test).

In two final studies mice received pv immunization and transplantationas before, but now also received iv injection with commercial anti-ratOX-2 (×4 injections; 100 mg/mouse at 2 day intervals). As shown in FIGS.5A and B and 6 these infusions of anti-OX-2 significantly decreased theprolonged graft survival (FIG. 6) and increased expression of OX-2antigen (Western blotting-FIGS. 5A and 5B) seen following pvimmunization. No perturbation of graft survival following pvimmunization was seen using additional treatments with anti-CD28/antiCTLA4 (see FIG. 6), or, in studies not shown, using anti-B7-1 oranti-B7-2. Again infusion of the IgG1 isotype control Mab (clone 107.3)did not alter the increased graft survival seen following pvimmunization (see FIG. 6).

In separate experiments cells were harvested from mice receiving pvimmunization along with additional treatment with monoclonal antibodiesas show (see Table 2). Following treatment with anti-OX-2 there was nolonger the altered cytokine production (with polarization to productionof IL-4 and IL-10) which the inventor has described in multiple modelsystems in which animals received pv donor-specific pre-transplantimmunization. Treatment with any of the other 4 monoclonal antibodiestested did not produce this reversal in polarization of cytokineproduction seen following pv immunization-indeed, using these Mabs alonein the absence of pv immunization produced a trend to increased graftsurvival (not shown) and significant polarization in cytokine productionto increased IL-4 and IL-10 production, akin to that produced by pvimmunization itself (upper half of Table 2).

OX-2 is a molecule previously characterized by Barclay et al. (1981,1982) as being preferentially expressed on rat thymocytes and dendriticcells. Dendritic cells are known to be important signalling cells forlymphocytes, which also potentially regulate cytokine production andgraft rejection, and infusion of dendritic cells is a potent means ofinducing pv tolerance. The inventor has determined that OX-2 expressionincreased following pv immunization, and further studied whether thishad any functional consequences. As shown in FIGS. 4 and 5, there isindeed significantly increased expression of OX-2 in spleen cellsisolated from pv immunized mice, along with the increased graft survivaland polarization in cytokine production (FIG. 6 and Table 2). Incontrast, in vivo infusion of anti-OX-2 abolishes increased expressionof this molecule, simultaneously reversing the increased graft survivaland altered cytokine profile seen. This data is consistent with thepossible function of OX-2⁺ cells in promoting allograft survival.

In the studies described the donor dendritic cells infused via theportal vein were themselves OX-2⁺ (see description of materials andmethods above). However, identical data in FACS analysis (FIG. 4) andWestern Blots (FIG. 5), and from suppression subtraction hybridization(FIG. 3), have been obtained in studies in which we used irradiatedwhole spleen cells (OX-2³¹ by FACS) for pv infusion. This is consistentwith the lack of evidence for increased mRNA expression of OX-2 early(1-2 days) post transplant, as noted above. Thus it seems most likelythat an operationally important “OX-2 signal” detected in the spleen ofthe pv immunized mice can derive from new expression, rather thannecessarily from infused OX-2⁺cells. In the absence of a polymorphicmarker for OX-2, however, it cannot be determined whether increasedexpression is from donor or host cells (or both). Indeed, it is perhapssomewhat surprising that the murine antibody to rat OX-2 cross-reacts inthe fashion shown with murine OX-2. Definitive analysis of the in vivorole of OX-2 awaits similar studies to those above, using antibodiesdeveloped against the murine OX-2 homologue-these experiments arecurrently in progress. It is also important to point out that while pvimmunization led to only a 4-fold alteration in the absolute number ofdetectable OX-2⁺ NLDC⁺ cells in the spleen/liver (see text and FIG. 4),nevertheless in the face of this 4-fold difference a clear difference inOX-2 signals in Northern gels using RNA from pv vs iv immunized mice(FIG. 3), along with evidence for a role for this quantitativedifference in the outcome of graft survival (FIG. 6) were detected.Presumably these results reflect respectively the limitation to thesensitivity of the Northern assay used, and some function of thequantitation of “co-stimulation” occurring after OX-2:OX-2 ligandinteraction.

While there was some 50% homology of the predicted protein sequence ofmurine OX-2 with murine B7-1, B7-2, CD28 and CTLA4 (Borriello et al.,1997), antibodies to the latter molecules did not reverse the prolongedgraft survival and altered cytokine production following pv immunization(FIG. 6, Table 2—see also (Castle et al., 1993)). In fact these latterantibodies themselves, infused in the absence of pv immunization,produced some of the same changes in cytokine production induced by pvimmunization (Table 2).

Example 2 Murine OX-2

This example describes the cloning and sequencing of murine MRC OX-2.

A cDNA library was constructed from MLN cells derived from adult C3Hmice, preimmunized 5 days earlier with 10×106 allogeneic B10.BR bonemarrow-derived dendritic cells allogeneic cells by the portal venous(pv) route, using a Cap Finder PCR cDNA library construction kit(Clontech). The inventor had previously isolated, using a PCR-SelectcDNA subtraction hybridization kit (Clontech) and RNAs obtained frompooled MLN of mice immunized by the pv route or via the lateral tailvein (iv), a 350 bp amplicon which showed over 98% homology with the 3′untranslated region of rat MRC OX-2 cDNA. Northern blot analysisconfirmed that this amplicon detected a differentially expressed productin RNAs prepared from iv vs pv immunized mice. This amplicon was used toscreen 5×105 clones of the amplified library. The sequences of cDNAclones were established with an Applied Biosystems 377 AutomatedSequencer, utilizing the Dye Terminator Cycle Sequencing method (AppliedBiosystems, Foster City, Calif.). The nucleotide sequence reported inthis paper has been submitted to the GenBank/EMBL Data Bank withaccession number AF004023.

The cDNA shown in FIG. 7 and SEQ.ID.NO.:1 has an open reading frame of837 base pairs, and a deduced amino acid sequence (FIG. 8 andSEQ.ID.NO.:2) of 248 amino acids, of which 30 represent a cleaved leadersequence. The predicted molecular weight of this, and the equivalentmolecules in rat and human, is approximately 25 kDa. The measuredmolecular weight in rat thymocytes, where the molecule is highlygylcosylated, is 47 kDa.

The murine MRC OX-2 shows some 92% and 77% homology overall at the aminoacid level with equivalent molecules in rat or human respectively. Asnoted for the rat molecule, the sequence from a 203-229 seems likely torepresent a membrane spanning domain (highly hydrophobic region), whilethe region from 229-248 is likely the intracytoplasmic region, with astretch of highly basic residues immediately C-terminal to position 229.Homology in the combined transmembrane and C-terminal regions with ratand human shows some 98% and 85% similarity respectively. As predictedfrom membership in the Ig supergene family, there are a number ofconserved Cys residues forming the disulphide bonds between β-strands ofIg-like domains, (21 and 91; 130 and 184 respectively); residue 91 waspreviously found to be the most highly conserved among members of theimmunoglobulin superfamily. Homology between the N-terminal Ig-domainwith rat and human, versus the next Ig-domain, is 88% and 82%, or 97and73% respectively. This relative concentration in variability between ratand mouse in the V-terminal Ig-domain may be more understandable whenthe ligand specificity for the molecules in these species is clarified.Note that the presumed extracellular portion of the molecule (1-202)contains a number of sites for N-glycosylation which are preservedacross species (44, 65, 73, 80, 94, 127, 130 and 151). This waspreviously reported for the rat cDNA sequence, and inferred from themeasured size of the expressed material in rat thymocytes.

The intracytoplasmic region of the molecules has no sequence identitywith known signaling kinases, nor does it have the well-describedconsensus sequence for the immunoreceptor tyrosine activation motif(ITAM: DXXYXXLXXXXXXXYDXL). In addition, it lacks typical SH2 or SH3domains to serve as “docking sites” for adapter molecules which might inturn co-opt other protein kinases in an activation cascade. Accordinglythe ligand-binding activity of the extracellular domains presumablyrepresent the biologically important region of the molecule. Somepossible functions attributable to ligand interaction with OX-2 can beinferred from other data in the literature. A homologous molecule,Ng-CAM, has been reported to bind a protein-tyrosine phosphatase viaN-linked oligosaccharide residues, and protein tyrosine phosphatases areknown to play a key regulatory role in immune responses. More recentlyALCAM, another adhesion molecule member of the Ig superfamily, the genefor which is located close to that for OX-2 on chromosome 3 in humans,has been shown to bind CD6 (a member of the scavenger receptor cysteinrich family, SRCR), and antibodies to CD6 may themselves play a role inregulating immune function.

Example 3 OX-2 Positive Cells Inhibit Type-1 Cytokine Production

The inventor has shown that hepatic mononuclear, non-parenchymal, cells(NPC) can inhibit the immune response seen when allogeneic C57BL/6dendritic cells (DC) are incubated with C3H spleen responder cells.Cells derived from these cultures transfer increased survival of C57BL/6renal allografts in C3H mice. The inventor also found that increasedexpression of OX-2 on dendritic cells was associated with inhibition ofcytokine production and renal allograft rejection. The inventor furtherexplored whether inhibition by hepatic NPC was a function of OX-2expression by these cells.

Fresh C57BL/6 spleen derived DC were cultured with C3H spleen respondercells and other putative co-regulatory cells. The latter were derivedfrom fresh C3H or C57BL/6 liver NPC, or from C3H or C57BL/6 mice treatedfor 10 days by intravenous infusion of human Flt3 ligand (Flt3L).Different populations of murine bone-marrow derived dendritic cells fromcultures of bone marrow with (IL-4+GM-CSF) were also used as a source ofputative regulator cells. Supernatants of all stimulated cultures wereexamined for functional expression of different cytokines (IL-2, IL-4,IFNγ, TGFβ). It was found that fresh C57BL/6 splenic DC induced IL-2 notIL-4 production. Cells from the sources indicated inhibited IL-2 andIFNγ production, and promoted IL-4 and TGFβ production. Inhibition wasassociated with increased expression of OX-2 on these cells, as definedby semi-quantitative PCR and FACS analysis. By size fractionation, cellsexpressing OX-2 were a subpopulation of NLDC145+ cells. This dataimplies a role for cells expressing OX-2 in the regulation of inductionof cytokine production by conventional allostimulatory DC.

Materials and Methods

Mice: Male and female C3H/HEJ and B10.BR (H-2^(k/k)), B10.D2 (H-2^(d/d))and C57BL/6 (H-2^(b/b)) mice were purchased from the Jacksonlaboratories, Bar Harbour, Me. Mice were housed 5/cage and allowed foodand water ad libitum. All mice were used at 8-12 weeks of age.

Monoclonal antibodies: The following monoclonal antibodies (Mabs), allobtained from Pharmingen (San Diego, Calif., USA) unless statedotherwise, were used: anti-IL-2 (JES6-1A12; biotinylated, JES6-5H4 );anti-IL-4 (11B11, ATCC; biotinylated, BVD6-24G2); anti-IFNγ (R4-6A2,ATCC; biotinylated XMG1.2); anti-IL-10 JES5-2A5; biotinylated SXC-1); PEanti-B7-1/B7-2 (Cedarlane Labs, Hornby, Ontario, Canada).

Rat anti-mouse OX-2 monoclonal antibodies were prepared byImmuno-Precise Antibodies Ltd. (Victoria, BC, Canada) followingimmunization of rats with a crude membrane extract of LPS stimulatedmurine DC, followed by fusion with a non-secreting rat myeloma parentcell line (YB2/3H1.P2.G11.16Ag.20). Hybridoma supernatants were screenedin ELISA using plates pre-coated with a 40-45 Kd preparation of DCextracts run on Western gels (Barclay, A. N. 1981. Immunology 44:727;Barclay, A. N., and H. A. Ward. 1982. Eur. J. Biochem. 129:447).Positive clones were re-screened using FACS analysis of CHO cellstransduced with a cDNA clone encoding full-length murine OX-2 (Chen, Z.,H. Zeng, and R. M. Gorczynski. 1997. BBA. Mol. Basis Dis. 1362:6-10).FITC-conjugated F(ab′)2 rabbit anti-rat IgG (non cross-reactive withmouse IgG) from Serotec, Canada was used as second antibody. The Mabselected for further analysis (M3B5) was grown in bulk in a CELLMAXsystem (Cellco Inc., Germantown, Md.). A crude preparation of ratimmunoglobulin (30% saturated ammonium sulphate preparation) was used asa control Ig.

In tissue culture assays where anti-cytokine Mabs were used to confirmthe specificity of the assay used 10 μg/ml of the relevant Mabs wasfound to neutralize up to 5.0 ng/ml of the cytokine tested.

NLDC145 (anti-mouse DC) was also obtained from Serotec. Recombinantmouse IL-4 was a kind gift from Dr. L. Yang (The Toronto Hospital);mouse rGM-CSF was purchased from Pharmingen. Recombinant human Flt3L(derived from CHO cells) was a kind gift from Dr. A. B. Troutt, ImmunexCorp., Seattle, Wash., USA.

Renal Transplantation

Renal transplantation was performed essentially as described elsewhere(Gorczynski, R. M. et al. 1994a. i Transplantation 58:816-820). Animalswere anesthetized with a combination of halothane and nitrous oxideinhalation, using novogesic for post-op analgesia. Orthotopic renaltransplantation was performed using routine procedures. In brief, Donoranimals received 200 Units of heparin, and kidneys were flushed with 2ml of ice cold heparinized physiological saline solution, prior toremoval and transplantation into recipient animals with leftnephrectomy. The graft renal artery was anastomosed to the recipient'sabdominal aorta, and the renal artery was anastomosed to the recipient'sinferior vena cava. The ureter was sewn into the recipient bladder usinga small donor bladder patch. All recipients received im injection withcefotetan (30 mg/Kg) on the day of transplantation and for 2 succeedingdays. The remaining host kidney was removed 2 days aftertransplantation, unless otherwise indicated. Treatment of recipientswith pv immunization, by monoclonal antibodies, or by oral immunizationwas as described in individual studies.

Portal Vein and Oral Immunization

Portal vein and oral immunization was performed as described earlier(Gorczynski, R. M. 1995a. Cell. Immunol. 160:224-231; Gorczynski, R. M.et al. Transplantation 62:1592-1600). All animals were anaesthetizedwith nembutal. A midline abdominal incision was made and the visceraexposed. Cells were injected in 0.1 ml through a superior mesentericvein using a 30 gauge needle. After injection the needle was rapidlywithdrawn and hemostasis secured without hematoma formation by gentlepressure using a 2 mm 3 gel-foam.

Bone-marrow derived dendritic cells (DC) for pv immunization wereobtained by culture of T depleted bone marrow cells in vitro with rIL-4and rGM-CSF (Gorczynski, R. M. et al. Transplantation 62:1592-1600).Staining with NLDC145 and FITC anti-rat IgG, or with FITC anti-CD3confirmed>95% NLDC145+ and <5% CD3+ cells at day 10 of culture(Gorczynski, R. M. et al. Transplantation 62:1592-1600). These cellswere washed and injected into mice or used for mixed leucocyte cultures.

Preparation of Cells:

Spleen and bone marrow (Gorczynski, R. M. et al. Transplantation62:1592-1600) cell suspensions were prepared aseptically from individualmice in each experiment. Hepatic mononuclear nonparenchymal cells (NPC)were isolated essentially as described elsewhere (Gorczynski, R. M.1994b. Immunology 81:27-35). Tissue was first digested at 37° C. for 45min with a mixture of collagenase/dispase, prior to separation (15 minat 17,000 rpm at room temperature) over mouse lymphopaque (CedarlaneLabs). Mononuclear cells were resuspended in a-Minimal Essential Mediumsupplemented with 2-mercaptoethanol and 10% fetal calf serum (aF10).Where cells were obtained from Flt3L injected mice, animals were treatedby iv injection of 10 mg/mouse Flt3L daily for 10 days. After enzymedigestion recovery of liver/spleen cells from these mice was markedlyincreased compared with saline-injected controls (120×10⁶, 390×10⁶ vs7×10⁶ and 120×10⁶ respectively).

Cytotoxicity and Cytokine Assays:

In cultures used to assess induction of cytotoxicity or cytokineproduction responder cells were stimulated with irradiated (2000R)stimulator cells in triplicate in αF10. Supernatants were pooled fromreplicate wells at 40 hrs for cytokine assays (below). No reproducibledifferences in cytokine levels have been detected from cultures assayedbetween 36 and 54 hrs of stimulation. In some experiments the culturesreceived 1 μCi/well (at 72 hrs) of ³HTdR and proliferation was assessedby harvesting cells 14 hrs later and counting in a well-type β-counter.

Where cytoxicity was measured cells were harvested and pooled fromequivalent cultures at 5 days, counted, and recultured at differenteffector:target with ⁵¹Cr EL4 (H2^(b/b)) or P815 (H₂ ^(d/d)) tumortarget cells. Supernatants were sampled at 4 hrs for assessment ofspecific cytotoxicity.

IL-2 and IL-4 activity were assayed by bioassay using the IL-2/IL-4dependent cell lines, CTLL-2 and CT4.S respectively. Recombinantcytokines for standardization of assays was purchased from Genzyme(Cambridge, Mass.). IL-2 assays were set up in the presence of 11B11 toblock potential stimulation of CTLL-2 with IL-4; IL-4 assays were set upin the presence of S4B6 to block IL-2 mediated stimulation. Both theIL-2 and IL-4 assays reproducibly detected 50 pg of recombinantlymphokine added to cultures.

In addition, IL-2, IL-4, IFNγ and IL-10 were assayed using ELISA assays.For IFNγ the assay used flat-bottomed Nunc plates (Gibco, BRL) coatedwith 100 ng/ml R4-6A2. Varying dilutions of supernatant were bound intriplicate at 4° C., washed×3, and biotinylated anti-IFNγ (XMG1.2)added. After washing, plates were incubated with streptavidin-horseradish peroxidase (Cedarlane Labs, Hornby, Ontario), developed withappropriate substrate, and OD₄₀₅ determined using an ELISA plate reader.Recombinant IFNγ for standardization was from Pharmingen. IL-10 wassimilarly assayed by ELISA, using JES5-2A5 as a capture antibody andbiotinylated SXC-1 as developing antibody. rIL-10 for standardizationwas from Pepro Tech Inc. (Rocky Hill, N.J.). Each assay detected 0.1ng/ml cytokine. ELISA assays for IL-2 and IL-4 used JES6-1A12 and 11B11as capture antibodies, with JAS6-5H4 or BVD6-24G2 as developingantibodies. Sensitivity of detection was 20 pg/ml for each cytokine.Where checked the correlation between bioassay and ELISA for IL-2 orIL-4 was excellent (r>0.90). In all studies reported below, data areshown from ELISA assays only. Where cytokine data are pooled fromseveral studies (e.g. FIGS. 14, 16, 17), absolute values of cytokineproduction were obtained as above using commercial recombinant cytokinesto standardize the assays. In our hands, supernatants fromC3Hanti-C57BL/6 cultures, under the conditions described, reproduciblycontain 950±200 and 80±25 pg/ml IL-2 and IL-4 respectively.

Preparation of RNA:

Different sources of tissue from renal-grafted female mice receiving DCand kidney allografts from male mice were harvested for RNA extractionas described elsewhere (Gorczynski, R. M. 1995a. Cell. Immunol.160:224-231). The OD280/260 of each sample was measured and reversetranscription performed using oligo (dT) primers (27-7858: Pharmacia,USA). The cDNA was diluted to a total volume of 100 ml with water andfrozen at −70° C. until use in PCR reactions with primers for murineGAPDH, B7-1, B7-2 or OX-2. The sense (S) and antisense (AS) primers weresynthesized by the Biotechnology Service Centre, Hospital for SickChildren, Toronto, using published sequences. 5′ primers were ³²Pend-labeled for PCR and had comparable levels of specific activity afterpurification by ethanol precipitation. 5 ml cDNA was amplified for 35cycles by PCR, and samples were analyzed in 12.5% polyacrylamide gelsfollowed by overnight (18 hrs) exposure for autoradiography. In controlstudies, using H-Y primer sets, this technique reliably detects H-Y mRNAfrom extracts of female spleen cells to which male cells are added at aconcentration of 1:105 (Gorczynski, R. M. 1995a. Cell. Immunol.160:224-231; Gorczynski, R. M. et al. Transplantation 62:1592-1600).Quantitative comparison of expression of different PCR products useddensitometric scanning of the autoradiograms.

(SEQ.ID.NO.:10) GAPDH Sense: 5′TGATGACATCAAGAAGGTGGTGAAG3′(SEQ.ID.NO.:11) GAPDH Antisense: 5′TCCTTGGAGGCCATGTAGGCCAT3′(SEQ.ID.NO.:12) B7-1 Sense: 5′CCTTGCCGTTACAACTCTCC3′ (SEQ.ID.NO.:13)B7-l Antisense: 5′CGGAAGCAAAGCAGGTAATC3′ (SEQ ID.NO.:14) B7-2 Sense:5′TCTCAGATGCTGTTTCCGTG3′ (SEQ.ID.NO.:15) B7-2 Antisense:5′GGTTCACTGAAGTTGGCGAT3′ (SEQ.ID.NO.:16) OX-2 Sense:5′GTGGAAGTGGTGACCCAGGA3′ (SEQ.ID.NO.:17) OX-2 Antisense:5′ATAGAGAGTAAGGCAAGCTG3′

Statistical Analysis:

In studies with multiple groups, ANOVA was performed to comparesignificance. In some cases (as defined in individual circumstances)pairwise comparison between groups was also subsequently performed.

Results

Antigen stimulation, in the presence of hepatic NPC, induces developmentof a cell population capable of inhibiting proliferation and IL-2production on adoptive transfer.

In a previous manuscript (Gorczynski, R. M. et al., Transplantation. 66:000-008) it was reported that C3H spleen cells stimulated in thepresence of syngeneic NPC and allogeneic (C57BL/6) DC produced a cellpopulation able to inhibit generation of IL-2 from fresh spleen cellsstimulated with C57BL/6 DC, and capable of inhibiting C57BL/6 renalallograft rejection in vivo. In order to ask whether this function ofNPC was MHC restricted or not, the following study was performed.

C57BL/6 (H2^(b/b)) spleen cells were stimulated in vitro with B10.BR(H2^(k/k)) bone-marrow derived DC, in the presence/absence of thefollowing NPC: C57BL/6; B10.BR; B10.D2 (H2^(d/d)). In addition, controlcultures were incubated with the NPC only. Proliferation and IL-2/IL-4production was measured in one aliquot of these primary cultures. Inaddition, at 5 days, cells were harvested from another set of theprimary cultures, washed, and 2×10⁵ cells added to cultures containing5×10⁶ fresh C57BL/6 spleen cells and BL10.BR DC. Proliferation andcytokine production was measured in these latter cultures in standardfashion. Data pooled from three equivalent studies are shown in panelsA) and B) of FIG. 9.

FIG. 9 is a bar graph showing regulation of proliferation and cytokineproduction following stimulation by allogeneic DC using hepatic NPC inaccordance with the methods described herein. In panel A) cultures wereinitiated with 5×10⁶ C57BL/6 responder spleen cells alone (group 1), orwith 2×10⁵ B10.BR DC (group 2). Further groups (3-5, and 6-8respectively) contained C57BL/6 responder cells and 2×105 NPC fromeither C57BL/6, B10.D2 or B10.BR respectively (3-5) or these same NPCand B10.BR DC (6-8). Data show mean proliferation and cytokineproduction from triplicate cultures in three separate studies. In panelB) data show proliferation and cytokine production from cultures of5×10⁶ C57BL/6 responder spleen cells stimulated in triplicate with 2×105B10.BR DC alone, or with the addition also of 2×10⁵ cells harvested fromthe cultures shown in the upper panel. Again data represent arithmeticmeans of 3 separate experiments. *p<0.05 compared with control cultures(far left in each panel).

There are a number of points of interest. As previously documented,addition of NPC syngeneic with spleen responder cells (C57BL/6 in thiscase) to cells stimulated with allogeneic (B10.BR) DC led to decreasedproliferation and IL-2 production from those responder cells comparedwith cells stimulated by DC alone (compare groups 6 and 2 of upper panelof FIG. 9, panel A). IL-4 production in contrast was enhanced. NPCalone, whether syngeneic or allogeneic to the responder cells, producedno obvious effect (groups 3-5, panel A) of FIG. 9). Furthermore, cellsfrom primary cultures receiving the DC+NPC mixture were able to inhibitproliferation and IL-2 production (while promoting IL-4 production) fromfresh spleen cells stimulated in secondary cultures with the same(B10.BR) DC (see panel B) of FIG. 9). However, data in this Figure makeanother important point. The same inhibition of proliferation/IL-2production in primary cultures was seen using either B10.BR NPC (MHCmatched with the DC stimulus-group 8, panel A) of FIG. 9) or withthird-party B10.D2 NPC (MHC-mismatched with both spleen responder cellsand allogeneic stimulator DC-group 7, panel A) of FIG. 9). Again noobvious effect was seen in cultures stimulated with B10.BR or B10.D2 NPCalone (groups 4 and 5). Finally, cells taken from primary culturesstimulated with DC and NPC from either B10.BR or B10.D2 could alsoinhibit proliferation/IL-2 production from secondary C57BL/6 spleen cellcultures stimulated with B10.BR DC-again cells taken from primarycultures with NPC alone produced no such inhibition (see panel B) ofFIG. 9). Thus the inhibition of proliferation/IL-2 production andenhancement of IL-4 production seen in primary cultures, as well as theinduction of suppression measured in secondary cultures, all induced byNPC, are not MHC-restricted.

Specificity of Inhibition/Suppression Induced by Hepatic NPC:

One interpretation of the data shown in FIG. 9 and elsewhere is that NPCdeliver a signal to DC-stimulated cells which is distinct from theantigen-signal provided by the DC themselves (and is MHCnon-restricted). This signal modulates the antigen-specific signalprovided by the DC. In order to assess the antigen-specificity of theimmunoregulation described in FIG. 9, the following experiment wasperformed.

C57BL/6 spleen responder cells were stimulated with B10.D2 or B10.BRbone marrow-derived DC, in the presence/absence of NPC from B10.BR orB10.D2 mice. Proliferation and cytokine production was measured inaliquots of these cultures as before. In addition, further aliquots ofcells harvested from these primary cultures were added to cultures offresh C57BL/6 spleen cells stimulated with B10.BR (panel B)—FIG. 10) orB10.D2 (panel C)—FIG. 10) DC. Again proliferation and cytokineproduction was measured. Data pooled from three such studies are shownin FIG. 10.

FIG. 10 shows specificity of inhibition of proliferation of cytokineproduction by hepatic NPC (see FIG. 9 and description of FIG. 9 for moredetails). In panel A), 5×106 C57BL/6 spleen cells were stimulated intriplicate for 3 days with 2×10⁵ B10.BR or B10.D2 DC, with/without 2×10⁵NPC derived from B10.D2 or B10.BR mice. Data shown are arithmetic meansof 3 repeat studies. In panels B) and C), fresh C57BL/6 responder spleencells were cultured in triplicate with either B10.BR DC (panel B), orB10.D2 DC (Panel C), with/without 2×10⁵ additional cells from theprimary cultures (groups 1-6 in panel A). Again data representarithmetic means of proliferation/cytokine production from 3 studies.*p<0.05 compared with control cultures (far left in each panel).

Data from the primary cultures (panel A)) recapitulates the observationsmade in FIG. 9, and show that NPC inhibit proliferation and IL-2production from DC-stimulated responder cells in an antigen andMHC-unrestricted fashion. However, the data in panels B) and C) of thisfigure show clearly that adoptive transfer of inhibition using cellsfrom these primary cultures occurs in an antigen-restricted fashion,dictated by the antigen-specificity of the DC used in the primarycultures, not of the NPC used for induction of suppression. Theseauxiliary cells in the NPC population thus have a functional property ofbeing “facilitator cells for induction of suppression”. Note that inother studies (data not shown) where the final assay system involvedmeasuring cytotoxicity to allogeneic target cells, a similar inhibitionof lysis (rather than cytokine production) was seen using cellsharvested from primary cultures stimulated with DC and hepatic NPC (seeGorczynski, R. M., et al. 1998a. Transplantation. 66: 000-008).

Hepatic Cell Preparations from Flt3L Treated Mice are a Potent Source ofDC and “Facilitator” Cells:

It has been reported at length that pv infusion of alloantigen, or ivinfusion of liver-derived allogeneic mononuclear cells inducesoperational unresponsiveness in recipient animals (Gorczynski, R. M.1995a. Cell. Immunol. 160:224-231; Gorczynski, R. M. et al.Transplantation 62:1592-1600; Gorczynski, R. M. et al. 1994a.Transplantation 58:816-820.; Gorczynski, R. M., and D. Wojcik. 1992.Immunol. Lett. 34:177-182; Gorczynski, R. M. et al. 1995b.Transplantation. 60:1337-1341). The total hepatic mononuclear cell yieldfrom normal mice is of the order of 5×10⁶ cells/mouse. In order toincrease the yield, and explore the possibility that the liver itselfmight be a source both of allostimulatory DC and “facilitator” cells 2C57BL/6 mice were exposed for 10 days to daily iv infusions of 10mg/mouse human CHO-derived Flt3L, a known growth factor for DC (Steptoe,R. J. et al. 1997. J Immunol. 159:5483-5491). Liver tissue was harvestedand pooled from these donors and mononuclear cells prepared as describedin the Materials and Methods section above (mean 130×10⁶ cells/donor).These cells were further subjected to sub-fractionation by size usingunit gravity sedimentation techniques (Miller, R. G., and R. A.Phillips. 1969. J. Cell. Comp. Physiol. 73:191-198). A typical sizeprofile for recovered cells is shown in FIG. 11 (one of 3 studies).

FIG. 11 shows OX-2 expression in a subpopulation of NPC. It is asedimentation analysis (cell profile) and FACS analysis of cellsisolated at 10 days from Flt3L-treated C57BL/6 mice. Two C57BL/67 micereceived 10 μg/mouse Flt3L iv daily for 10 days. Hepatic NPC weresedimented for 3 hrs at 4° C., and the fractions shown collected (Fxs1-4 with sedimentation velocities 2.5-3.8, 3.8-5.1, 5.1-6.4 and 6.4-8.0mm/hr respectively). Aliquots of the cells were stained in triplicatewith the Mabs shown. The remainder of the cells were used as in FIGS.12-14. Data are pooled from 3 studies.

In these same studies cells isolated from the various fractions shown inFIG. 11 were tested as follows. Firstly, cells were stained withFITC-labeled Mabs to B7-1, B7-2, NLDC145 and rat anti-mouse OX-2 (M3B5)with FITC anti-rat IgG as second antibody. In addition, mRNA extractedfrom the different cell samples were assayed by PCR for expression ofGAPDH, B7-1, B7-2 and OX-2. Data are shown in FIGS. 11 (pooled from 3separate studies) and FIG. 12 (representative PCR data from oneexperiment).

FIG. 12 shows PCR detection of B7-1, B7-2 and OX-2 in hepatic NPMC. Itis a PCR analysis for mRNA expression of OX-2, B7-1 and B7-2 in varioushepatic NPC cell fractions isolated from Flt3L treated mice (see FIG.11). Data are representative from 1 of 3 studies.

Further aliquots of the cells were used to stimulate fresh C3H spleenresponder cells in culture. Proliferation and cytokine assays wereperformed as before (see FIG. 9), and in addition cells were taken fromthese primary cultures and added to fresh secondary cultures of C3Hspleen responder cells and C57BL/6 bone marrow-derived DC. Againproliferation and cytokine production was assayed from these secondarycultures. Data pooled from 3 studies of this type are shown in FIG. 13(panels A) and B).

FIG. 13 shows that hepatic NPMC from Flt3L treated mice results IL-2 andIL-4 production. Stimulation of proliferation/cytokine production by NPCfrom Flt3L treated mice, and inhibition of the same (where stimulationis induced by a separate population of DC) is a function of differentcell populations. (See text and FIGS. 11-12 for more details.) HepaticNPC fractions were derived from Flt3L treated C57BL/6 mice and were usedto stimulate C3H spleen cells in triplicate cultures, alone or in thepresence of bone-marrow derived C57BL/6 DC (see panel A). Data showarithmetic means for proliferation/cytokine production from 3experiments. In addition, cells harvested from these primary cultureswere added to fresh C3H spleen cells stimulated with C57BL/6 DC (panelB), and again proliferation/cytokine production assayed. *p<0.05compared with control groups (far left of panel).

Finally, cells from the various fractions were infused iv into 2/groupC3H mice which also received C57BL/6 renal allografts as antigenchallenge. Spleen cells were harvested from these individual mice 10days after transplantation and restimulated in culture with C57BL/6 orB10.D2 DC, again with cytokines measured at 40 hrs (see FIG. 14).

FIG. 14 is a bar graph of cytokines produced from cells from C3H micewith C57BL/b renal allografts and NPC from Flt3 treated C57BL/6 donors.OX-2⁺ NPC infused iv into renal transplant allograft recipients leads topolarization of cytokine production (to IL-4, IL-10 and TGFβ) in spleencells harvested from those mice and restimulated in vitro. Fractions ofNPC from Flt3L treated C57BL/6 mice (from FIG. 11) were infused iv into2/group C3H recipients, receiving C57BL/6 renal allografts (along withCsA) in standard fashion (see Materials and Methods). Mice weresacrificed 14 days after transplantation and spleen cells stimulated invitro in triplicate with C57BL/6 DC stimulator cells. Cytokines wereassayed in the supernatants of these cultures at 60 hrs. Data showarithmetic means pooled from cultures in 3 studies of this type. *p<0.05compared with control groups (far left-no NPC infused).

Data in FIG. 11 show that distinct subpopulations of slow-sedimentingcells express OX-2 in the cells harvested from Flt3L treated mice, whencompared with cells expressing B7-1 and/or B7-2. In general expressionof OX-2 and B7-2 occurred in equivalent subpopulations.Faster-sedimenting cells (Fx 3 and 4 in FIG. 11), while staining forNLDC145, were positive by fluorescence mainly for B7-1, not B7-2 orOX-2. Similar conclusions were reached both by FACS analysis of cellpopulations (FIG. 11), and by PCR analysis of mRNA (FIG. 12).

When the functional capacity of these different cell populations wasinvestigated (FIGS. 13 and 14) it was found that optimal directstimulation (or proliferation and IL-2 production) was seen from B7-1expressing cells (Fxs 3 and 4 in panel A) of FIG. 13), while only OX-2expressing cells (Fxs 1 and 2 in FIGS. 11 and 12) were capable ofproducing the inhibitory effects defined earlier (FIGS. 9 & 10) in thetwo-stage culture system (panel B) in FIG. 13). These same cells (Fxs 1and 2) were in turn able, after iv infusion, to polarize cells from micegiven renal allografts to produce predominantly IL-4, IL-10 and TGFβproduction on restimulation in vitro (FIG. 14). These data areconsistent with the notion that after FltL treatment of mice expansionof a population of immunostimulatory DC occurs within the liver, whichalso contains another distinct population of (facilitator) cells whichpromote immunoregulation.

Evidence that Cell Populations with “Facilitator” Activity from theLiver of Flt3L Treated Mice Prolong Graft Survival in vivo:

Since it has been reported elsewhere that there is a good correlationbetween treatments (such as pv immunization) which decrease IL-2production and increase IL-4 production from restimulated cells andprolongation of graft survival (Gorczynski, R. M., and D. Wojcik. 1994.J. Immunol. 152:2011-2019; Gorczynski, R. M. 1995a. Cell. Immunol.160:224-231; Gorczynski, R. M. et al. Transplantation 62:1592-1600), andthat increased expression of OX-2 is also independently associated withincreased graft survival after pv immunization (Gorczynski, R. M. et al.1998b. Transplantation. 65:1106-1114), the next question was whethercells isolated from Flt3L treated mice which induced inhibitory functionin vitro (see FIGS. 9, 10 and 13), and expressed increased amounts ofOX-2 (FIGS. 11, 12) were themselves capable of promoting increased graftsurvival in vivo.

Groups of 2 C57BL/6 mice received iv infusions of 10 mg/mouse Flt3L for10 days as before. Cells were isolated from the liver by enzymedigestion, and fractionated by unit gravity sedimentation. 4 pools ofcells were recovered, and an aliquot stained as before in FACS withanti-OX-2. Groups of 2 C3H mice received 10×10⁶ cells iv from the 4separate pools. A control group received saline injections only. Overthe next 48 hrs all mice received C57BL/6 renal transplants. All micereceived CsA (10 mg/Kg) on the day of renal transplantation. Data inFIG. 15 are pooled from 3 studies of this type (representing 6mice/group), and show the animal survival in these 5 different groups.

FIG. 15 shows NPC from Flt3L treated C57BL/6 mice, infused iv intorecipient C3H mice, inhibit C57BL/6 renal allograft rejection. Two micegroups received the different subpopulations of NPC derived from Flt3Ltreated mice shown in FIGS. 11 and 12. Fxs 1 and 2 were OX-2⁺. Micereceived C57BL/6 renal allografts within 48 hrs along with CsA (seeMaterials and Methods). Animal survival was followed as an end point.Data shown are pooled from 3 studies (6 mice/group). *p<0.05 comparedwith mice receiving CsA only (n).

It is quite clear from this FIG. that only hepatic cells expressing OX-2(Fxs 1 and 2—see FIGS. 11 and 12) were capable of promoting increasedgraft survival after iv infusion. Comparison of these data with those inFIG. 13 confirm that these cell populations were also those identified,using a 2-stage culture assay system, as cells with functional“facilitator” activity (see also FIGS. 9 and 10). There was nosignificant difference in survival between groups receiving NPC-Fx1 orNPC-Fx2 in this experiment, in keeping with relatively equivalent levelsof OX-2 expression in these fractions (FIG. 11).

Anti-OX-2 Monoclonal Antibody in vitro Reverses Regulation Induced byHepatic NPC:

A final study was directed to whether anti-OX-2 monoclonal antibodyM3B5, added to cultures of C3H spleen responder cells, allogeneic(C57BL/6) DC and NPC from C57BL/6 mice, could prevent the inhibition ofIL-2 production in primary cultures, and the development of cells ableto inhibit such cytokine responses from freshly stimulated respondercells in secondary cultures (see FIGS. 9, 10 and 13). Data in FIGS. 16and 17 are pooled from 3 studies of this type.

FIG. 16 is a bar graph showing the effect of anti B7-1; B7-2; or OX-2 onprimary allostimulation. It shows that anti-OX-2 Mab increases IL-2cytokine production in vitro after stimulation of C3H responder spleencells with C57BL/6 DC. Subgroups of cultures contained the Mabs shown.Cytokines were assayed at 60 hrs. All data represent arithmetic meanspooled from 3 repeat studies. *p<0.05 compared with control group (farleft).

FIG. 17 is a bar graph showing that anti-OX-2 reverses inhibition byNPC. It shows that anti-OX-2 Mab inhibits development ofimmunoregulatory cells in vitro following incubation with hepatic NPC.C3H responder spleen cells were incubated in triplicate with C57BL/6 DCalong with NPC (see FIGS. 9 and 10). Subgoups of these culturescontained the Mabs shown. Cytokines were assayed in cultures at 60 hrs(panel A). In addition, cells were harvested from all groups, washed andadded to fresh C3H responder spleen cells and C57BL/6 DC (panel B).Cytokines in these groups were assayed 60 hrs later. All data representarithmetic means pooled from 3 repeat studies. *p<0.05 compared withcontrol group from cultures of NPC with no monoclonal antibodies (farleft in Figure)—see also FIG. 16.

Primary cultures were of two types, containing C3H responder spleencells and C57BL/6 DC alone (FIG. 16), or the same mixture with addedC57BL/6 NPC (FIG. 17). Subsets of these cultures contained in additioneither 5 μg/ml of anti-B7-1, anti-B7-2 or anti-OX-2. Supernatants fromresponder cells stimulated in the presence of DC only were assayed after60 hrs for cytokine production (FIG. 16). For the primary culturesincubated with both DC and NPC, supernatants were harvested at 60 hrsand tested for cytokine production (FIG. 17A). In addition, cells wereharvested after 5 days, washed, and added to secondary cultures of freshC3H responder cells with fresh C57BL/6 DC. No monoclonal antibodies wereadded at this second culture stage. Data for cytokine production thesesecondary cultures are shown in FIG. 17B.

Addition of anti-B7-1 or anti-B7-2 to DC stimulated spleen cultures ledto inhibition of cytokine production (FIG. 16), while in contrastanti-OX-2 monoclonal antibody led an increase in IL-2 production inthese primary cultures (FIG. 16). We have reported similar findingselsewhere (Ragheb et al-submitted for publication). Interestingly,anti-OX-2 abolished the inhibition of cytokine production caused by NPCin these primary cultures (FIG. 17A—see also FIGS. 9, 10 and 13). Inaddition, anti-OX-2 prevented the functional development of a cellpopulation capable of transferring inhibition of cytokine production tofreshly stimulated spleen cells (FIG. 17B).

Discussion

There is considerable theoretical as well as practical interest inunderstanding the mechanism(s) by which a state of antigen specifictolerance can be induced in lymphoid populations. Limits to theeffective induction of tolerance represent a major challenge to moresuccessful allo (and xeno) transplantation, to name but one example(Akatsuka, Y., C. Cerveny, and J. A. Hansen. 1996. Hum. Immunol.48:125-134). Significant efforts have been invested into exploring howpre- (or peri-) transplant donor-specific immunization might producesuch a state (Qian, J. H. et al. 1985. J. Immunol. 134:3656-3663;Kenick, S., et al. 1987. Transpl. Proc. 19:478-480; Gorczynski, R. M.1992. Immunol. Lett. 33:67-77; Thelen, M., and U. Wirthmueller. 1994.Curr. Opin. Immunol. 6:106-112; Akolkar, P. N. et al. 1993. J. Immunol.150 (April 1):2761-2773; Ahvazi, B. C. et al. J. Leu. Biol. 58(1):23-31; Albina, J. E. et al. 1991. J. Immunol. 147:144-152). There isgood evidence that portal venous (pv) immunization somehow leads totolerance induction, and this immunoregulation can apparently bemonitored by following changes in cytokine production from host cells,with decreased production of IL-2, IL-12 and IFNγ, and increased IL-4,IL-10, IL-13 and TGFβ ( Thelen, M., and U. Wirthmueller. 1994. Curr.Opin. Immunol. 6:106-112; Gorczynski, R. M. et al. 1998a.Transplantation. 66: 000-008). Which, if any, of these cytokine changesis directly and causally implicated nevertheless remains obscure.

Further analysis of the cell population able to induce tolerance afterpv immunization led to the somewhat paradoxical observation that donordendritic (DC) cells represented an excellent tolerizing population(Gorczynski, R. M. 1995a. Cell. Immunol. 160:224-231; Gorczynski, R. M.et al. Transplantation 62:1592-1600). Since antigen-pulsed DC areconventionally thought of as representing an optimal immunizing regime,the mechanism(s) activated following DC pv immmunization which led totolerance (Banchereau, J., and R. M. Steinman. 1998. Nature.392:245-252) was of interest. It is already clear that DC themselvesrepresent an extremely heterogeneous population, in terms of origin,cell surface phenotype, turnover in vivo and possibly function (Salomon,B. et al. 1998. J. Immunol. 160:708-717; Leenen, P. J. M. et al.1998. 1. Immunol. 160:2166-2173). In the mouse lymph node at least 3discrete populations were identified, one of which comprised smallCD8α⁺NLDC145⁺cells, likely of lymphoid origin, with an immaturephenotype, and whose numbers were profoundly increased (100×) followingFlt3L treatment in vivo (Salomon, B. et al. 1998. J. Immunol.160:708-717) (administration of the latter has been reported to lead toproliferation of dendritic cells and other cells of hematopoietic origin(Maraskovsky, E. et al. 1996. J. Exptl. Med. 184:1953-1962)). Thesecells resembled the interdigitating DC found in the T cell areas of thesplenic white pulp, and have been implicated in regulation of immunityinduced by other (myeloid derived) DC (Salomon, B. et al. 1998. J.Immunol. 160:708-717; Kronin, V. et al. 1996. J. Immunol. 157:3819-3827;Suss, G., and K. Shortman. 1996. J. Exptl. Med. 183:1789-1796).

A variety of other studies have indicated that the induction of immunity(vs tolerance) following antigen presentation was intrinsicallydependent upon the co-existence of other signaling ligands at thesurface of DC (interacting with appropriate counter-ligands on thesurface of other cells (e.g. stimulated T cells)) (Larsen, C. P. et al.1994. J. Immunol. 152:5208-5219; Lenschow, D. J. et al. 1996. Annu. Rev.Immunol. 14:233-258; Larsen, C. P., and T. C. Pearson. 1997. Curr. Opin.Immunol. 9:641-647). It was speculated that infusion of DC via theportal vein induced tolerance by co-opting another cell population,distinguishable by expression of unique cell surface ligands, whosebiological function was to facilitate induction of tolerance, notimmunity, when antigen was presented in association with otherwiseimmunogenic DC. Some preliminary evidence supporting this hypothesis wasrecently reported (Gorczynski, R. M. et al. 1998a. Transplantation. 66:000-008). Herein, this is referred to as a facilitator cell. Moreover,because pv immunization has been shown to be associated with increasedexpression of a novel molecule, OX-2, previously reported to beexpressed on DC (Barclay, A. N. 1981. Immunology 44:727; Barclay, A. N.,and H. A. Ward. 1982. Eur. J. Biochem. 129:447; Chen, Z. et al. 1997.BBA. Mol. Basis Dis. 1362:6-10; Gorczynski, R. M. et al. 1998b.Transplantation. 65:1106-1114), it was predicted that this moleculewould in fact serve as a “marker” for the hypothetical facilitator celldescribed. Experiments reported herein are consistent with such ahypothesis.

It is here shown that within the hepatic NPC population there is asubset of cells able to inhibit stimulation by allogeneic DC in anon-MHC restricted fashion (see FIGS. 9 and 10), and able to induce thedevelopment of an antigen-specific immunoregulatory cell population invitro (see FIGS. 9 and 10). The non-MHC-restricted nature of this“facilitator” cell interaction indicates that it functions by providingan accessory signal (a regulatory not a co-stimulatory signal) to the DCwhich stimulate T cells in the allogeneic mixed leukocyte reactiondescribed, in a fashion analogous to the original description ofcostimulatory interactions (Jenkins, M. K. et al. 1988. J. Immunol.140:3324-3329). As a result the stimulated lymphocytes alter theircytokine production profile (with decreased IL-2 production andproliferation), and become able to regulate the immune response seenfrom freshly stimulated lymphocytes (see panel B in FIGS. 9 and 10).Most interestingly, following expansion of DC in vivo by Flt3Ltreatment, it is shown that in fact the liver itself contains both animmunostimulating population (large cells by velocity sedimentationanalysis), and this putative “facilitator” cell population (see FIGS.11-15). Furthermore, the latter biological activity resides within aslow-sedimenting (small size) NLDC145⁺ cell population expressingpreferentially both cell surface B7-2 and OX-2 (see FIGS. 11 and 12).When it was investigated whether this same population of cells wasactive in vivo in regulating graft tolerance, it was found again thatafter prior Flt3L treatment, the liver contained a population of cellswhich transferred increased renal graft acceptance (FIG. 15) and inparallel altered the cytokine production profile of immunized micetowards increased IL-4 and TGFβ, and decreased IL-2 and IFNγ production(FIG. 14).

In a final attempt to explore the role for OX-2 expression itself inthis regulatory function, fresh spleen cells were stimulated with DCalone or in the presence of anti-B7-1, anti-B7-2 or anti-OX-2. Note thatother studies (data not shown) have confirmed that even the bone-marrowderived DC used contains small numbers of OX-2⁺ cells (RMG-unpublished).Unlike anti-B7-1 and anti-B7-2 which decreased cytokine production, aresult in keeping with the hypothesized role for these as costimulatormolecules (Hancock, W. W. et al. 1996. Proc. Natl. Acad. Sci. USA.93:13967-13972; Freeman, G. J. et al. 1995. Immunity. 2:523-532;Kuchroo, V. K. et al. 1995. Cell. 80:707-718), anti-OX-2 produced asmall but significant (1.7-2.5 fold in three studies) increase in IL-2production in this system (FIG. 16). Most important, however, inclusionof anti-OX-2 Mab in a system where exogenous “facilitator” cells wereadded (from NPC), blocked completely the induction of inhibitionnormally seen in such cultures (FIGS. 9 and 10; compare with lower panelof FIG. 17). These data are consistent with the concept that OX-2delivers a regulatory, not a costimulatory, signal in this situation.

How does the present data fit within the evolving framework ofunderstanding in the heterogeneity of DC? As noted above, there has beenspeculation that a separate population of CD8α⁺NLDC145⁺ DC of lymphoidorigin which proliferates in response to Flt3L, might be responsible forimmunoregulation. Other data have implicated IL-10 as a cytokine whichmight modify development/maturation of DC into a population expressingincreased amounts of B7-2 and capable of inducing tolerance (Steinbrink,K. et al. 1997. J Immunol. 159:4772-4780). The role of regulation ofexpression of Fas as a controlling feature in this regard is unexplored(Suss, G., and K. Shortman. 1996. J. Exptl. Med. 183:1789-1796). Thedata disclosed herein is the first to implicate another molecule, OX-2,in the delivery of a tolerizing signal, perhaps in association withalterations in expression of B7-2, Fas etc. It is intriguing that whilethere is clearly a key role for intra-thymic DC in the regulation ofself-tolerance (Banchereau, J., and R. M. Steinman. 1998. Nature.392:245-252), natural expression of OX-2 was initially first describedon thymic DC (as well as within the brain) (Barclay, A. N. 1981.Immunology 44:727)—there is as yet no evidence to suggest that thisrepresents a functionally relevant expression for OX-2 in this location.However, other independent data have also implied an immunoregulatoryrole for OX-2 expression, again as assayed by altered cytokineproduction in vitro from cells stimulated in the presence/absence ofexpressed OX-2 (Borriello, F. et al. 1997. J. Immuno.. 158:4548).

It has been reported that following pv immunization there is ameasureable expansion in numbers of populations of γδTCR⁺ cells capableof adoptive transfer of increased graft survival to naive recipients(Gorczynski, R. M. et al. 1996c. Immunology. 87 (3):381-389). Little isknown concerning the nature of the antigen recognized by these cells,and why, as a population, their numbers are preferentially increasedfollowing pv immunization. It is speculated that this may be explainableultimately in terms of a differential susceptibility of γδTCR⁺ vs αβTCR⁺cells to immunoregulatory signals delivered following OX-2 expression.

In conclusion, the inventor has reported for the first time thatfunctional heterogeneity in the DC pool may be understandable in termsof differential expression of OX-2 on the cell surface. Expression ofthis molecule seems to give cells the capability to induceimmunoregulation, increased renal graft survival (and altered cytokineproduction both in vivo and in vitro). The present invention suggeststhat such OX-2 expressing cells are referred to as “facilitator” cells(for tolerance induction).

Example 4 Preparation of Murine Antibodies

Mouse and rat hybridomas to a 43 Kd molecule expressed in the thymus, ona subpopulation of dendritic cells, and in the brain, in mammaliantissue derived from mouse, rat and human were prepared. Using CHO cellstransiently transfected with adenovirus vector(s) expressing a cDNAconstruct for the relevant OX-2 gene, the monoclonal antibodies (Mabs)detect a molecule encoded by this construct (rat OX-2 (rOX-2), mouseOX-2 (mOX-2) and human OX-2 (huOX-2) respectively). Furthermore, atleast some of the anti-rat Mabs detect determinants expressed on themurine OX-2 molecule.

Material and Methods

Antigen preparation from tissues and Western blotting were performed asdescribed in Gorczynski et al., Transplantation, 1998, 65:1106-1114:

Spleen cells (human samples were obtained from cadavers at the time oforgan retrieval for transplantation) were used for preparation ofdendritic cells/macrophages. Tissue was digested with a mixture ofcollagenase and dispase and centrifuged over lymphopaque. Cells wereadhered for 2 hr at 37° C., washed vigorously, and incubated for 14 hrat 37° C. Dendritic cells were isolated as non-adherent cells(Gorczynski et al., Transplantation, 1996. 62:1592-1600). Routinestaining of mouse splenocytes with NLDC-145 and FITC anti-rat IgG, orFITC-MAC-1 before and after overnight incubation produced the followingstaining pattern in these adherent cells: 8%±2%, 90%±11% and 92%±9%,9%±3% respectively. The crude (non-adherent) dendritic cell preparationwas extracted with lysis buffer, titred to a protein concentration of 10mg/ml, and used for immunization. Some of the same material was usedsubsequently in screening ELISAs (below).

When brain tissue was used in Western gel analysis, whole tissue extractwas electrophoresed in 12%SDS-PAGE and transferred to PVDF membranes(Novex Co., San Diego, Calif.). Putative anti-OX-2 Mabs were used astest reagent, with isotypic antibodies (negative in ELISA tests) used ascontrols. Membranes were developed using either anti-rat or anti-mousehorse radish peroxidase and appropriate substrate.

Immunization and Production of Mabs:

Four female BALB/c mice were initially immunized by intraperitonealinjections with 1 mg of human or rat dendritic antigen in CompleteFreundis Adjuvant. Three subsequent boosts were administered as above,spaced at 3 week intervals, with Incomplete Freundis Adjuvant. When theserum titre had risen more than 10-fold from a pre-immune serum sample,as determined by ELISA, the 2 highest responders were boostedintravenously. Three days later the donor mice were sacrificed and thespleen cells were harvested and pooled. Fusion of the splenocytes withX63-Ag8.6.5.3 BALB/c parental myeloma cells was performed as previouslydescribed (Kohler, G. and C. Milstein. 1975. Nature. 25: p. 256-259),except that one-step selection and cloning of the hybridomas wasperformed in 0.8% methylcellulose medium (Immuno-Precise AntibodiesLtd., Victoria, BC). This proprietary semi-solid medium allows HATselection and cloning in a single step and eliminates the overgrowth ofslower growing desirable clones by faster growing, perhaps undesirable,hybridomas. Clones were picked and resuspended in wells of 96-welltissue culture plates in 200 μl of MEM medium containing 1%hypoxanthine/thymidine, 20% Fetal Bovine serum, 1% OPI, and 1×106/mlBALB/c thymocytes. After 4 days, the supernatants were screened by ELISAfor antibody activity on plates coated with the immunizing antigen.Putative positive hybridomas were re-cloned by limited dilution cloningto ensure monoclonality and screened in FACS on extracts prepared frombrain tissue (below).

For the production of rat mAbs, 2 Fisher rats were immunized as abovewith mouse antigen. Essentially the same procedure was followed, exceptthe parental cell line used for the fusion was YB2/0.

ELISA and FACS Analysis of Putative Mabs:

ELISA assays used polystyrene plates pre-coated with 100 ng/mlpoly-L-lysine, followed by overnight incubation with the crude dendriticcell antigen (used for immunization) at 10 mg/ml. Wells were developedafter binding of hybridoma superntatants using the anti-rat/anti-mousehorse radish peroxidase antibodies above and plates were analysed in anautomatic ELISA plate reader (TiterTek Multiskan, MCC/340, FlowLabs,Mississauga, Ontario, Canada).

FACS analysis was performed using putative anti-OX-2 Mabs and thefollowing cells. Fresh peripheral blood leucocytes (PBL), isolated overrat/mouse lymphopaque (Cedarlane laboratories) or Ficoll-Hypaque(human); fresh spleen dendritic cells (isolated after adherence andovernight incubation, as above); and CHO cells transduced with viralvectors engineered to contain a single copy of a cDNA inserted into thenot1/bamH1 sites, encoding the relevant species-specific OX-2, as perpublished sequences (Chen, Z. et al. 1997. BBA. Mol. Basis Dis.1362:6-10; McCaughan, G. W., et al. 1987. Immunogenetics. 25: p.133-135), or with control vector alone. FITC anti-mouse (or anti-rat)IgG was used as secondary antibody.

Mixed Leucocyte Reactivity (MLR) and Cytokine Production:

Allogeneic MLR cultures, using 1:1 mixtures of 2.5×10⁶ responder PBL andmitomycin C treated stimulator PBL, were set up in 24-well cultureplates in 1 ml of aMEM medium supplemented with 10% FCS. Cells wereobtained from C3H responder mice (with stimulator C57L/6), Lewis (LEW)rats (with Brown Norway, BN, as stimulator), and individual humandonors. Culture supernatants were harvested at 60 hrs and tested fordifferent cytokines using previously described ELISA assays (mouse), orusing CTLL-2 as bioassay for IL-2 production from all responder cellsources (Gorczynski, R. M., et al. 1998c. Immunology. 93: p. 221-229).

Results

Evaluation of a Number of Mabs for Staining of Cell Populations in FreshPBL or Spleen:

All Mabs tested in the experiments herein described were previouslyscreened as described in the Materials and Methods above, and detected amolecule in Western gel of brain extracts with Molecular Weight 42-45Kd, and also stained CHO transduced by OX-2 encoding viral vectors. Datain Table 3 show FACS analysis for these Mabs using fresh cells. The dataare summed over several independent analyses, using a number of Mabsdirected to rat, mouse or human OX-2, for staining of cells harvestedfrom fresh PBL or spleen (adherent cells only were tested for the latterthese represented some 5-8% of the total cell population in all cases).

It is clear from Table 3 that PBL in all species tested contained some1.3%-2.5% OX-2⁺ cells by FACS analysis, and that spleen adherent cellssimilarly contained 4%-8% OX-2⁺ cells. As confirmation of the inventor'sprevious work, spleen adherent cells taken from C3H mice or LEW ratstreated 4 days earlier by portal venous immunization with 20×106 (or50×10⁶ respectively) of C57BL/6 (or BN) bone marrow cells showed some3.5-5 fold elevation in OX-2⁺ cells (see Table 3). Under theseconditions specific increases in survival of subsequentallo-transplanted cells/tissue have been reported (Gorczynski, R. M. etal. 1996a. Transplantation 62:1592-1600).

Ability of Anti-OX-2 Mabs to Modulate Cytokine Production in MLR invitro:

In a final study the issue of whether these Mabs can modify the immuneresponse (as assayed by cytokine production) of cells stimulated in anallogeneic mixed leucocyte reaction (MLR) in vitro was addressed. Theinventor has previously shown that cells taken from mice pretreated byportal allogeneic immunization produce predominantly type-2 cytokines,and that an anti-OX-2 Mab could apparently reverse this polarization incytokine production (and indeed abolish the increased graft survivalseen in such mice). Data in Table 4 confirm these results using 3independent Mabs to mouse OX-2. Further, rat or human cells stimulatedin the presence of anti-rat (or human) OX-2, similarly show morepronounced IL-2 production than cells stimulated in the presence ofisotypic control Ig (or no Ig), without a generalized increase incytokine production (as analysed here by no change in IL-6 production inany group).

Discussion

In the data in this example it is confirmed that using species specificMabs, to human, rat or mouse OX-2, that Mabs to the molecule detected onthe surface of host dendritic cells may play a role in regulatingcytokine production after allostimulation in vitro, and moreparticularly that functionally blocking OX-2 expression leads toenhanced IL-2 production (a type-1 cytokine) after allostimulation(Table 4). Borriello et al also recently reported that OX-2 expressionwas not a costimulator for induction of IL-2 and IFNγ synthesis(Borriello, F. et al. 1997. J. Immuno. 158:4548)—our data imply it is infact a negative signal for type-1 cytokine production. In micepreimmunized by the portal vein, as reported earlier, there is a 4-foldincrease in OX-2 expressing cells in PBL and spleen, and a reversal ofpolarization in cytokine production (from type2 cytokines to type-1cytokines) after stimulation of cells in the presence of OX-2 (seeTables 3 and 4) (Gorczynski, R. M. et al. 1998b. Transplantation.65:1106-1114).

Example 5 Preparation of Rat Antibodies

Five rats were immunized using GERBU adjuvant (GERBU Biotechnik,Gaiberg, Germany) with 500 μg of membrane protein purified from themouse dendritic cell (DC) line DC2.4 (a gift from K. Rock, Harvard).Serum from these rats was tested 7 days after the third immunization,and compared with a pre-immunization sample in an ELISA usingplate-bound material of Mol. Wt. 40 Kd-45 Kd eluted from Western blots,and Alk Pase anti-rat Ig. Two rats with high titre antibody werere-immunized and sacrificed 4 days later for fusion of spleen cells withHAT-sensitive Sp2/0 parent cells for preparation of hybridomas.Hybridomas were screened by ELISA (56/960+ve), subcloned, and frozen(−70° C.). For further specificity testing of the anti-OX-2 Mabs willuse CHO cells can be transfected with a pBK eukaryotic expression vector(Stratagene,Calif.) expressing OX-2. Full length OX-2 cDNA, includingthe leader sequence, was amplified from DC2.4 cells using sense andantisense primers constructed with Spe1 or Xba1 sites respectively attheir 5′ ends for directional cloning into the vector. A band of theexpected size (849 bp) was obtained on agarose gel electrophoresis. Thesequence of the cloned cDNA was confirmed by sequencing using anautomated DNA sequencer (Chen, Z. and Gorczynski, R. M. 1997. Biochem.Biophys. Acta. 100, in press). CHO cells were transfected byelectroporation (5×106 cells in 0.5 ml were pulsed at 960 MH₂ and 120Vusing a Bio-Rad Gene Pulser (Bio-Rad, Hercules, Calif.), using the fulllength OX-2 expression plasmid along with a plasmid encoding puromycinresistance (100:1 ratio), followed by selection in puromycin (12 μg/mlfor 4 days). Puromycin resistant cells were cloned by limiting dilution.5 CHO transfectant clones have been obtained expressing mRNA for OX-2 asconfirmed by PCR. These clones can be used to screen the putative ratanti-mouse OX-2 Mabs.

(a) FACS Staining of Cells From pv Immunized Mice with Anti-mouse OX-2

A 4-fold increase in staining of spleen and hepatic NLDC145+ (dendriticcell marker) cells from pv immunized mice with anti-rat OX-290 wasobserved. Spleen and liver tissue of mice at various times (12 hours; 2,7 and 14 days) following pv immunization can be sectioned and stained byimmunohistochemistry, using anti-NLDC145, anti-OX-2 Mabs. Single cellsuspensions from the same tissues can be stained, using 3-colour FACS,with FITC-anti-mouse OX-2, rhodamine-anti-NLDC145, andphycoerythrin-anti-T200 (mouse lymphocyte marker). In all cases (bothFACS and immunohistochemistry) the appropriate irrelevant isotypecontrol antibodies are included. Tissue from control mice receivingrenal grafts alone, or following additional iv immunization, can also beexamined. Detection of NLDC145+ (and/or MAC-1+) cells showing increasedexpression of OX-2 is predicted in pv immunized mice only (seeGorczynski, R. M. et al. 1998. J. Immunol. 160, in press). The inventorhas shown DC-associated antigen persists only in animals with survivinggrafts (Gorczynski, R. M., Chen, Z., Zeng, H. and Fu, X. M. 1998.Transplantation submitted). It was also assessed whether anti-OX-2,infused at different times post transplantation, causes rejection (b).

(b) Modulation of Graft Rejection and Cytokine Production by Anti-mouseOX-2

C3H mice receive pv immunization with cultured C57BL/6 bone-marrowderived dendritic cells (DC), CsA and renal allografts. Groups of micereceive intravenous infusion of various rat anti-mouse OX-2 Mabs(100-500 μg/mouse, ×5, at 2 day intervals), beginning at different timespost transplantation (this will be guided by data from (a)). Serumcreatinine and animal survival are followed. Serum from Mab-treated miceare tested in ELISA and by FACS with OX-2 expressing CHO transfectants(above) to ensure antibody excess. If OX-2 expression is important forpv induced increased graft survival, the anti-OX-2 treated pv immunizedmice will reject grafts like untreated controls, with similarpolarization of cytokine production to type-1 cytokines (assayed by PCR;ELISA with cultured, restimulated cells). As controls pv immunized,grafted mice receive anti-CD28 and anti-CTLA4 these Mabs do not modifythe effects of pv immunization as assayed by graft survival orpolarization in cytokine production. It is expected that OX-2 treatmentbut not other Mabs, will simultaneously abolish expansion of γδTCR+cells after pv immunization.

Example 6 Preparation of a Fusion Protein Linking the ExtracellularDomain of OX-2 to Mouse Fc

Immunoadhesins, in which a hybrid molecule is created at the cDNA levelby fusing the extracellular domain (ED) of an adhesion molecule with thecarboxyl terminus of IgG heavy chain, the whole being expressed inmammalian cells or in a baculovirus system, have been powerful tools inthe identification and isolation of the counter ligands for the adhesionmolecule of interest. Ligands for a number of members of the TNFRfamily, were identified in this fashion (Goodwin, R. G. et al. 1993.Eur. J. Immunol. 23,2631-2641; Gruss, H. and Dower, S. 1995. Blood 85,3378-3404). Interest has developed in the potential application ofimmunoadhesins as therapeutic agents. A CTLA4 immunoadhesion, with thecapacity to bind both B7-1 and B7-2, has been used to inhibit T cellcostimulation and decrease rejection (Larsen, C. P. et al. 1996. Nature381, 434-438). Note that CD28/CTLA4 are not counter ligands for OX-289.The fusion protein, is predicted to alter cytokine production (increasedIL-4, IL-10; decreased IL-2, IFNγ) and increase renal graft survivallike pv immunization. We expect that synergistic blockade ofcostimulation (e.g. by CTLA4-Fc) and triggering of a coregulatorypathway (by OX-2ED-Fc) will induce tolerance and produce indefinitegraft survival.

a) Construction of an OX-2 Fusion Protein With Murine IgGFc2a

A cDNA encoding the extracellular region of OX-2 (OX-2ED) was amplifiedby PCR, using a 5′ oligonucleotide primer which inserts a Sal1 site 5′immediately at the start of the V-region sequence and a 3′ primer whichcreates a BamH1 site at the 3′ end (the site of junction with Fc). UsingcDNA prepared from mouse ConA activated spleen cells, with a 5′ primercontaining an Spe1 site, and a 3′ primer containing a Sal1 site, thesignal peptide for IL-6 (SP-IL-6) was amplified by PCR and ligated tothe OX-2ED amplicon. In frame ligation across the junction of SP-IL-6and OX-2ED was checked by manual sequencing-the final cDNA amplified bythe 5′SP-IL-6 primer and the 3′OX-2ED primer was, as expected, 695 bp. Aplasmid expressing murine IgGFc2a (Fcγ2a), modified to create a uniqueBamH1 site spanning the first codon of the hinge region, and with aunique Xba1 site 3′ to the termination codon, has been obtained from Dr.Terry Strom (Zheng, X. X. et al. 1995. Journal of Immunology. 154,5590-5600). The IgGFc2a in this insert has been further modified toreplace the C1 q binding motif (rendering it non-lytic) and inactivatethe FcγR1 binding site (see Zheng, X. X. et al. 1995. Journal ofImmunology. 154, 5590-5600). Ligation of OX-2ED and IgGFc2a in thecorrect reading frame at the BamH1 site yields a 1446 bp long openreading frame encoding a single 478-amino acid polypeptide (includingthe 24-amino acid IL-6 signal peptide). The homodimer has a predicted105 kDa Mol Wt, exclusive of glycosylation. The fusion gene is thencloned as an Spe1-Xba1 cassette into the eukaryotic expression plasmidpBK/CMV (Stratagene, CA). This plasmid has a CMV promoter/enhancer and aneomycin-resistance gene for selection using G418. The appropriategenetic construction of the OX-2ED-Fc can be confirmed by directsequencing after cloning into the plasmid vector (Chen, Z. andGorczynski, R. M. 1997. Biochem. Biophys. Acta. 100, in press)—see alsoabove. The plasmid is transfected into CHO cells by electroporation (seeabove), and selected in medium with 1.5 mg/ml G418 (Geneticin:LifeTechnologies, Inc.). After subcloning, high producing clones areselected by screening culture supernatants in ELISA using anti-OX-2 Mabsas capture antibody, and Alk Pase coupled anti-IgGFc2a as detectionantibody. OX-2ED-Fc fusion protein is purified from culture supernatantsusing protein A-Sepharose affinity chromatography, dialysed against PBS,filter-sterilized and stored in aliquots at −20° C. The size, and OX-2(+IgGFc2a) specificity of the secreted product can be confirmed usingWestern blot analysis under reducing (+DTT) and non-reducing (−DTT)conditions, with Mabs to OX-2 and rat monoclonal anti-mouse IgGFc2a(Pharmingen). The product can be titrated as an inhibitor for FACSstaining of OX-2 expressing CHO cells (see above) using rat Mabs to OX-2as probe. As a prelude to studies (below) using OX-2EDFc in vivo, thehalf-life (t½) in mouse serum following injection of groups of 6 8-weekC3H mice will be studied. This is carried out by subjecting mice to ivinjections of 50 μg or 10 μg of OX-2ED-Fc, and obtains serial 50 μlblood samples at 0.3, 1, 6, 24, 48, 72 and 96 hours. The serum isanalyzed in ELISA using plates coated with anti-OX-2 as captureantibody, and Alk Pase coupled monoclonal anti-IgGFc2a for detection(thus ensuring the assay detects only OX-2ED-Fc, not OX-2 or IgGFc2aalone). Based on earlier data in which Fc fusion proteins were used toextend the in vivo half-life, a t½in the range of 30-40 hrs (Zheng, X.X. et al. 1995. Journal of Immunology. 154, 5590-5600) is predicted.

b) OX-2: IgGFc Immunoadhesion Inhibits MLR

CHO cells were transduced with a vector carry the OX-2:Fc cDNA insert.Supernatant was harvested from the CHO cells at 7 days and was culturedwith 5×10⁶ LEW spleen and 2.5×10⁶ irradiated LBNFI spleen cells. Thesupernatant contained 50 ng/ml OX-2:Fc.

The results, shown in Table 5, demonstrate that the soluble OX-2:Fcimmunoadhesion inhibits IL-2 production and generation of cytotoxic Tcells and induces IL-4 production. These results support the use of OX-2as an immunosuppressant.

c) Use of OX-2:Fc in vivo for Prevention of Graft Rejection

It was shown in (b) that incubation in the presence of 50 ng/ml OX-2:Fccan inhibit an in vitro MLR reaction. To detect inhibition of in vivograft rejection, C3H mice received C57BL/6 skin grafts along with ivinjection of OX-2:Fc (50 μg/mouse) every 2 days ×4 injections. Graftswere inspected daily after 10 days for rejection. In a separate study 3mice/group (receiving saline or OX-2:Fc) were sacrificed at 10 days andspleen cells restimulated in vitro (×48 hrs) for analysis of cytokineproduction. Data for these studies is shown in Tables 6 and 7. It isclear from these data that OX-2:Fc has the potential for use as animmunosuppressant to prolong graft acceptance. Furthermore, inassociation with increased graft survival in this model, OX-2:Fc alterspolarization in cytokine production, as already described for portalvein donor-specific immunization.

Example 7 OX-2 Prevents Fetal Loss

Using in situ hybridization, the inventor has shown that OX-2 is notexpressed in the placenta of mice with increased potential for fetalloss. In contrast, OX-2 is expressed in the placenta of normal,non-aborting mice.

CBA/J and DBA/2J mice were used. Matings of CBA/J(females) with DBA/2Jmales show a high incidence of fetal loss (>80%), unlike the reversescenario. Placental tissue was obtained from matings at 8-11 days ofgestation. Uteri were snap frozen, 5 μm sections cut, and stained with abiotinylated anti-sense probe for murine OX-2. Data shown in FIGS. 18Aand 18B indicate increased expression of OX-2 mRNA (in situ labeling) inthe non-aborting strain combination, with essentially absent expressionin the aborting combination. These data are consistent with the notionthat OX-2 expression prevents spontaneous fetal loss syndrome.

While the present invention has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

TABLE 1 Summary of sequences and clones detected in cDNA library from pvimmunized mice Match category Number of clones represented (%) Knownmouse genes 30 (45) Non-mouse genes (rat/human) 14 (21) No data basematch 22 (34) Footnotes: Genes were considered a “match” with a BLASTscore >250 with a minimum of 50 bp alignment.

TABLE 2 Cytokine production from cells of mice receiving pv immunizationand anti-rat OX-2 Mabs given Cytokine levels in culture supernatants^(b)to recipients^(a) IL-2 IFNγ IL-4 IL-10 No pv immunization (CsA only)None 750 ± 125  85 ± 18 29 ± 8  130 ± 40  +anti-rat OX-2 890 ± 160  93 ±19 30 ± 10 120 ± 35  +anti-mouse CD28 415 ± 88*  57 ± 9* 105 ± 22* 275 ±55* +anti-mouse CTLA4  505 ± 125* 65 ± 8  95 ± 20* 190 ± 45  +anti-B7-1340 ± 65*  35 ± 7* 120 ± 21* 285 ± 60* +anti-B7-2 495 ± 90* 64 ± 7  90 ±20* 185 ± 45  PV immunization + CsA None 190 ± 55  25 ± 8 107 ± 21  780± 150 +anti-rat OX-2  730 ± 140*  60 ± 16*  33 ± 10* 220 ± 40*+anti-mouse CD28 145 ± 38  20 ± 9 145 ± 34  1140 ± 245  +anti-mouseCTLA4 85 ± 25 15 ± 6 125 ± 31  960 ± 220 +anti-B7-1 110 ± 30  20 ± 6 144± 28  885 ± 180 +anti-B7-2 75 ± 20 14 ± 5 150 ± 30  1230 ± 245 Footnotes: ^(a)3 C3H mice/group were used in each experiment. Allanimals received CsA and C57BL/6 renal transplants as described in theMaterials and Methods. Mice in the lower half of the Table also receivedpv infusions of 15 × 10⁶ C57BL/6 bone marrow derived dendritic cells onthe day of transplantation. Where monoclonal antibodies were given thedose used was 100 mg/mouse, ×4 doses at 2 day intervals. All mice weresacrificed 14 days post transplantation. Spleen cells were cultured intriplicate from individual animals for 40 hrs in a 1:1 mixture withirradiated C57BL/6 spleen stimulator cells. ^(b)Arithmetic mean (±SD)for triplicate determinations from individual samples of the animalsdescribed in the first coiumn. All cytokines were assayed by ELISA.IL-2, IL-4 and IL-10 are shown as pg/ml, IFNg as ng/ml. Data are pooledfrom 2 such studies (total of 6 individual mice tested/group).*represents significantly different from control group with no Mab (p <0.02)

TABLE 3 FACS staining of PBL and spleen adherent cells in differentspecies, using anti-OX-2 Mabs Donor^(b) Percent stained cells^(c)SPECIES^(a) Treatment Mab PBL Spleen Human NONE H4B4 1.5 ± 0.3 4.8 ± 1.7H4A9A2 1.5 ± 0.4 6.1 ± 2.0 H4A9C7 1.3 ± 0.4 4.3 ± 1.7 Mouse NONE M3B51.9 ± 0.4 6.7 ± 2.1 M3B6 1.7 ± 0.4 5.2 ± 1.6 M2C8 1.4 ± 0.4 4.2 ± 1.4Mouse PV immune M3B5 5.9 ± 1.5  20 ± 4.1 M3B6 5.2 ± 1.4  17 ± 3.6 M2C84.7 ± 1.4  15 ± 3.3 Rat NONE RC6A3 1.3 ± 0.3 5.3 ± 1.6 RC6C2 1.5 ± 0.46.5 ± 1.7 RC6D1 1.9 ± 0.6 6.8 ± 1.5 Rat PV immune RC6A3 4.8 ± 1.3  16 ±4.2 RC6C2 4.9 ± 1.6  18 ± 3.9 RC6D1 5.3 ± 1.7  20 ± 4.5 Footnotes:^(a)Fresh cells were obtained from normal human donors (PBL), cadaverictransplant donors (human spleen), or from adult (8-10 week) mouse or ratdonors. The same 3 separate tissue donors were used for each Mab tested.^(b) Donor pretreatment refers to infusion of allogeneic bone marrowcells into the portal vein (C57BL/6 for C3H mouse donors; BN for LEW ratdonors) 4 days before harvest of PBL or spleen (see tekt and (6)).^(c)Arithmetic mean (±SD) for percent cells stained in 3 independentassays. Control antibodies (FITC anti-mouse IgG (for anti-human oranti-rat Mabs, or FITC anti-rat IgG for anti-mouse Mabs) gave nosignificant staining above background (<0.2%).

TABLE 4 Type-1 cytokine production in MLR cultures is increased byanti-OX-2 Mabs Cytokine levels in culture supernatants^(b) ELISA assays(murine only) Bioassay (CTTL-2) Mabs in culture^(a) IL-2 IFNγ IL-4 IL-10IL-2 IL-6 MOUSE MLR None 350 ± 55  35 ± 18 345 ± 63  340 ± 50  480 ±160  365 ± 74 M3B5 890 ± 160* 115 ± 29* 130 ± 10* 168 ± 42* 820 ± 200*265 ± 46 M3B6 915 ± 155* 117 ± 25* 135 ± 32* 135 ± 38* 850 ± 175* 303 ±55 M2C8 855 ± 155* 105 ± 28* 120 ± 32* 140 ± 37* 830 ± 165* 279 ± 61control Ig 370 ± 75  36 ± 11 330 ± 55  310 ± 45  335 ± 60  349 ± 59None** 710 ± 145  108 ± 23  110 ± 21  105 ± 23  690 ± 155  285 ± 54 RATMLR None 490 ± 145  360 ± 57 RC6A3 690 ± 155* 295 ± 55 RC6C2 845 ± 180*345 ± 68 RC6D1 830 ± 160* 370 ± 57 Control Ig 475 ± 160  356 ± 58 HUMANMLR None 395 ± 85  295 ± 45 H4B4 570 ± 125* 315 ± 50 H4A9A2 630 ± 145*320 ± 48 H4A9C7 625 ± 140* 345 ± 56 Control Ig 360 ± 120  320 ± 50Footnotes: ^(a)MLR cultures were set up as described in the Materialsand Methods. For human MLR cultures the same 3 different responderpreparations were used for each Mab, and stimulated with a pool ofmitomycin C treated spleen stimulator cells (from a random mixture of 6spleen donors). For mouse (C3H anti-C57BL/6) and rat (LEW anti-BN) MLRcultures all assays were set up in triplicate for each Mab. Mouseresponder spleen cells were from mice treated 4 days earlier by portalvein infusion of # C57BL/6 bone marrow cells, except for data shown as(None**) where responder cells were from non-injected C3H mice. Mab wasadded as a 30% superntatant concentration. Supernatants were harvestedfor cytokine assays at 60 hrs. ^(b)Data show arithmetic means (+SD) foreach Mab. For mouse assays all supernatants were assayed for a number ofcytokines (ELISA), and for IL-2/IL-6 using bioassays (proliferation ofCTLL-2, B9 respectively). Supernatants from rat/human cultures wereassayed in bioassays only. Note that cells incubated with isotypecontrol Igs (non-reactive by ELISA or FACS) gave cytokine dataindistinguishable from cultures incubated in the absence of Mab. p <0.05, compared with cultures # without Mabs.

TABLE 5 OX-2:FC Immunoadhesin Inhibits Mixed Leukocyte Reaction in vitroPercent lysis ⁵¹Cr targets^(b) Cytokines in culture (pg/ml)^(c) Addedsupernatant^(a) (50:1, effector:target) IL-2 IL-4 NONE (control)  31 ±4.0 1005 ± 185 60 ± 20 Control CHO  33 ± 4.3  810 ± 190 45 ± 20 (vectortransduced) CHO transduced with 4.2 ± 2.1 175 ± 45 245 ± 55  OX-2:FcFootnotes: ^(a)Supernatant was harvested at 7 days from CHO cellstransduced with control pbK vector, or vector carrying a cDNA insertencoding OX-2 linked to murine Fc. A 1:1 mixture of supernatant was usedin cultures containing 5 × 10⁶ LEW spleen and 2.5 × 10⁶ irradiated LBNF1spleen cells; this corresponded to 50 ng/ml OX-2:Fc ^(b) and ^(c)Percentlysis with cells at 5 days, using 1 × 10⁴ ⁵¹ Cr BN spleen ConA targets;cytokines in culture supernatants at 60 hrs.

TABLE 6 Inhibition of skin graft rejection by OX-2:Fc Treatment of miceRejection of skin grafts (mean + SD) in days NTL 12 + 3.8 OX-2:Fc 19 +4.2 Footnotes: 6 mice/group were treated as shown. NIL indicatesinfusion of normal mouse IgG only. Arithmetic mean (+SD) graft survivalfor group.

TABLE 7 OX-2:Fc infused into mice receiving skin allografts reversespolarization in cytokine production Cytokines in culture supernatant at48 hrs (pg/ml) Treatment of mice IL-2 IL-4 NIL 1250 + 160  80 + 20OX-2:Fc 350 + 85 245 + 50 Footnotes: 3 mice/group received iv infusionsof saline or OX-2:Fc (50 mg/mouse) every 2 days ×4 from the time ofgrafting with C57BL/6 skin. Mice were sacrificed at 10 days and spleencells stimulated in vitro with irradiated C57BL/6 spleen stimulatorcells. Arithmetic mean (+SD) for IL-2/IL-4 in supernatant at 48 hrs.Data are pooled from triplicate cultures for each mouse spleen.

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                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 22 <210> SEQ ID NO 1 <211> LENGTH: 2791<212> TYPE: DNA <213> ORGANISM: Mus musculus <400> SEQUENCE: 1actatagggc acgcgtggtc gacggcccgg gctggtactg agaaggaata gg#atgcagtc     60agagggaagg gacttgagga agacctttgg tttagactct ctccacatgt ct#gtctgtgg    120gtctctgaac cagattttat ctgttgctgc ctctctgatg acagctggtc aa#ggccccaa    180tctattagta tagcagaatg tcattaagaa tcattctttt cttccttcca tt#ttttcttc    240ttcttctact ccctccccct ttctctctct ctctttcttt ctttctttct tt#ctttttct    300ttctttcttt ctctctctcc ctctttctct ctctccctct ctctctttct tt#ctttcttt    360ctttctcttt ttctttctgt ctttctctct ctttccttct ttccttcccc ag#ctgtgttt    420ggttctaccc taggactctg ggctttccat tatctggttc ttgaccatcc ag#gcaatata    480ggtacaagct ctctcttata gggtgggcct caagttaaac taaacattgg tt#ggtcactc    540cctcacgttt tctactaaaa tctcataggc aggacatatt gtgggtagag ga#tttagagg    600caggtttagt gtccaggttt gtcttttcat ggtctgtaga ataccttctc ac#accagaga    660gactagagtc tagagtccaa acctcagctc tagcctctct atgttcagtg ag#ctgaatga    720aagttgacct cagcaatggg tcccactgtc aggttttaga gggtgacctt ca#gttgtagg    780tcccaagtct ctctctcctc tctctccctt tctcgatctc tctctctctc tc#tctctctc    840tctctctctc tctctctctc tctctctctc tctctctgct ttatacttgt ga#ttgaagat    900gtgatctctc tggcagcctg gtaccatgcc tcctggtcac ttagagactc tc#ctcctgta    960gctataagcc caacaaatct ttttccacag gtttctactc tagtacagaa ac#agaaatgt   1020caccaatata gtcaatcgtt tctgtaaagc tttcatcaag gaaaacctca gt#tccagggc   1080ttcctgtgac tcatttgatc tgtcccttga ttctcatctg ttttaaggaa ta#ctgcggga   1140caatctgatt agcagaaaga aagtgctttt gggttttcag gaagtgtgtt ca#caggtagc   1200tctgagccct taggacttct aaagctctag atgaggtacc tggtaaccac ac#acacacac   1260acacacacac acacacacac acacgcactg gcctttaata taacaaatca ta#aaataaag   1320tttttctttt tttttcccca gggtgtctgt atgaatctcc ttaccttctt cc#ccctacac   1380acacacacac acacacacac acacacacac acactattgt tctgttctcc ga#gtttacct   1440tttgctgtac agaaccacag gatgcaccgg gtttctgact caaattactg tc#cactcaag   1500ttagttccca ctccgatttt tctgtatgga ctacgtcacc ctatactgcc at#ttggcacg   1560ggagagaggc cagtgatggg aatgcagacg aaacatgcat acacatgtaa aa#taagataa   1620ataaatctaa aatgaaaaaa aatatagagt gattctttca catttttgct at#attactct   1680aaaaggcgag aacctggcgg gggcgggggc aggggctagg gacgaggttg ta#gagggcgt   1740ggttggttgg tcgtctcttc ctccacacta gaggagctgt agagtctgcc tg#tgcggtgg   1800agggggctct ctctacggcg aatagtagtg tccctgctca caggtgttgc gg#agatatcc   1860tccatcgtgg aagagctcag accccgagaa gctggtgtct agctgcggcc cc#gagcaagg   1920atgggcagtc tggtgagtgg aatctgagat gcgaaggagg gcggaatggg cg#atctggag   1980ccgcggctct cagaagccag tggagcctgc gagaaaagca aggaagctgt tc#tttggaga   2040agtggtatcc ggggctcgga gctctgtaag gaggcaccgg ccggagaaag cc#cggggaac   2100gcgtgtatct agggtgggcg gctttgctcc ttgctgcgat tccattgcga aa#acacggcc   2160tgagctccat ggctcccaga aggggaggag tagctctttg cgtcccctat gt#tggtcctt   2220aacctgcagc aggggtgtag cctagtaatc tcgcttgctc tctttctcac cc#cctctctt   2280gctgcatttc tgctccttgc ctagaaaacc atgaagcatc tagcagtact gc#agcgagca   2340agccacagct tagtggtctt gttaaatgcc aaggtattta gaggagaggc cg#acattttg   2400agtctttggt actgtttaca aggcagaaaa ttttaaaagg aagggtggtc at#acgcctta   2460ttctttatac acacggaatt ggtagaattg aatgcgaatc taaacgcaat ta#aaccccag   2520gtaccacttt tcatcaggct gacaaagacc gacttgtgtt acctttccta ac#aaagagga   2580atgtggatct gtcagctaga tgctcttagt gttcaaacaa ggaattgctt tc#tgttttac   2640aaagaatcgg agagagaggt tctttttttt ctctccaagt ctctgtggct gc#aatgaaat   2700aaggtacaaa atcagaccta gaaagaatag gggaatgggg ctatgcacct ag#cagaccag   2760 cccgggccgt cgaccacgcg tgccctatag t        #                   #        2791 <210> SEQ ID NO 2 <211> LENGTH: 278<212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 2Met Gly Ser Leu Val Phe Arg Arg Pro Phe Cy #s His Leu Ser Thr Tyr1               5    #                10   #                15Ser Leu Ile Trp Gly Met Ala Ala Val Ala Le #u Ser Thr Ala Gln Val            20       #            25       #            30Glu Val Val Thr Gln Asp Glu Arg Lys Ala Le #u His Thr Thr Ala Ser        35           #        40           #        45Leu Arg Cys Ser Leu Lys Thr Ser Gln Glu Pr #o Leu Ile Val Thr Trp    50               #    55               #    60Gln Lys Lys Lys Ala Val Ser Pro Glu Asn Me #t Val Thr Tyr Ser Lys65                   #70                   #75                   #80Thr His Gly Val Val Ile Gln Pro Ala Tyr Ly #s Asp Arg Ile Asn Val                85   #                90   #                95Thr Glu Leu Gly Leu Trp Asn Ser Ser Ile Th #r Phe Trp Asn Thr Thr            100       #           105       #           110Leu Glu Asp Glu Gly Cys Tyr Met Cys Leu Ph #e Asn Thr Phe Gly Ser        115           #       120           #       125Gln Lys Val Ser Gly Thr Ala Cys Leu Thr Le #u Tyr Val Gln Pro Ile    130               #   135               #   140Val His Leu His Tyr Asn Tyr Phe Glu Asp Hi #s Leu Asn Ile Thr Cys145                 1 #50                 1 #55                 1 #60Ser Ala Thr Ala Arg Pro Ala Pro Ala Ile Se #r Trp Lys Gly Thr Gly                165   #               170   #               175Thr Gly Ile Glu Asn Ser Thr Glu Ser His Ph #e His Ser Asn Gly Thr            180       #           185       #           190Thr Ser Val Thr Ser Ile Leu Arg Val Lys As #p Pro Lys Thr Gln Val        195           #       200           #       205Gly Lys Glu Val Ile Cys Gln Val Leu Tyr Le #u Gly Asn Val Ile Asp    210               #   215               #   220Tyr Lys Gln Ser Leu Asp Lys Gly Phe Trp Ph #e Ser Val Pro Leu Leu225                 2 #30                 2 #35                 2 #40Leu Ser Ile Val Ser Leu Val Ile Leu Leu Va #l Leu Ile Ser Ile Leu                245   #               250   #               255Leu Tyr Trp Lys Arg His Arg Asn Gln Glu Ar #g Gly Glu Ser Ser Gln            260       #           265       #           270Gly Met Gln Arg Met Lys         275 <210> SEQ ID NO 3 <211> LENGTH: 14<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Primer <400> SEQUENCE: 3ttttgtacaa gctt               #                   #                  #     14 <210> SEQ ID NO 4 <211> LENGTH: 44 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Adapter 1 <400> SEQUENCE: 4ctaatacgac tcactatagg gctcgagcgg ccgcccgggc aggt    #                  # 44 <210> SEQ ID NO 5 <211> LENGTH: 43 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Adapter 2 <400> SEQUENCE: 5tgtagcgtga agacgacaga aagggcgtgg tgcggagggc ggt     #                  # 43 <210> SEQ ID NO 6 <211> LENGTH: 22 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Primer 1 <400> SEQUENCE: 6ctaatacgac tcactatagg gc            #                  #                 22 <210> SEQ ID NO 7 <211> LENGTH: 22 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Nested Primer 1 <400> SEQUENCE: 7tcgagcggcc gcccgggcag gt            #                  #                 22 <210> SEQ ID NO 8 <211> LENGTH: 21 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Primer 2 <400> SEQUENCE: 8tgtagcgtga agacgacaga a            #                  #                   #21 <210> SEQ ID NO 9 <211> LENGTH: 22<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Nested Primer 2 <400> SEQUENCE: 9agggcgtggt gcggagggcg gt            #                  #                 22 <210> SEQ ID NO 10 <211> LENGTH: 25 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: GADPH Sense <400> SEQUENCE: 10tgatgacatc aagaaggtgg tgaag           #                  #               25 <210> SEQ ID NO 11 <211> LENGTH: 23 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: GADPH Antisense <400> SEQUENCE: 11tccttggagg ccatgtaggc cat            #                  #                23 <210> SEQ ID NO 12 <211> LENGTH: 20 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: B7-1 Sense <400> SEQUENCE: 12ccttgccgtt acaactctcc             #                  #                   # 20 <210> SEQ ID NO 13 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: B7-1 Antisense <400> SEQUENCE: 13cggaagcaaa gcaggtaatc             #                  #                   # 20 <210> SEQ ID NO 14 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: B7-2 Sense <400> SEQUENCE: 14tctcagatgc tgtttccgtg             #                  #                   # 20 <210> SEQ ID NO 15 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: B7-2 Antisense <400> SEQUENCE: 15ggttcactga agttggcgat             #                  #                   # 20 <210> SEQ ID NO 16 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: OX-2 Sense <400> SEQUENCE: 16gtggaagtgg tgacccagga             #                  #                   # 20 <210> SEQ ID NO 17 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: OX-2 Antisense <400> SEQUENCE: 17atagagagta aggcaagctg             #                  #                   # 20 <210> SEQ ID NO 18 <211> LENGTH: 825<212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 18gtgatcagga tgcccttctc tcatctctcc tcctacagcc tggtttgggt ca#tggcagca     60gtggtgctgt gcacagcaca agtgcaagtg gtgacccagg atgaaagaga gc#agctgtac    120acacctgctt ccttaaaatg ctctctgcaa aatgcccagg aagccctcat tg#tgacatgg    180cagaaaaaga aagctgtaag cccagaaaac atggtcacct tcagcgagaa cc#atggggtg    240gtgatccagc ctgcctataa ggacaagata aacattaccc agctgggact cc#aaaactca    300accatcacct tctggaatat caccctggag gatgaagggt gttacatgtg tc#tcttcaat    360acctttggtt ttgggaagat ctcaggaacg gcctgcctca ccgtctatgt ac#agcccata    420gtatcccttc actacaaatt ctctgaagac cacctaaata tcacttgctc tg#ccactgcc    480cgcccagccc ccatggtctt ctggaaggtc cctcggtcag ggattgaaaa ta#gtacagtg    540actctgtctc acccaaatgg gaccacgtct gttaccagca tcctccatat ca#aagaccct    600aagaatcagg tggggaagga ggtgatctgc caggtgctgc acctggggac tg#tgaccgac    660tttaagcaaa ccgtcaacaa aggatattgg ttttcagttc cgctattgct aa#gcattgtt    720tccctggtaa ttcttctcat cctaatctca atcttactgt actggaaacg tc#accggaat    780 caggaccgag gtgaattgtc acagggagtt caaaaaatga cataa   #                 825 <210> SEQ ID NO 19 <211> LENGTH: 274<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 19Val Ile Arg Met Pro Phe Ser His Leu Ser Th #r Tyr Ser Leu Val Trp1               5    #                10   #                15Val Met Ala Ala Val Val Leu Cys Thr Ala Gl #n Val Gln Val Val Thr            20       #            25       #            30Gln Asp Glu Arg Glu Gln Leu Tyr Thr Thr Al #a Ser Leu Lys Cys Ser        35           #        40           #        45Leu Gln Asn Ala Gln Glu Ala Leu Ile Val Th #r Trp Gln Lys Lys Lys    50               #    55               #    60Ala Val Ser Pro Glu Asn Met Val Thr Phe Se #r Glu Asn His Gly Val65                   #70                   #75                   #80Val Ile Gln Pro Ala Tyr Lys Asp Lys Ile As #n Ile Thr Gln Leu Gly                85   #                90   #                95Leu Gln Asn Ser Thr Ile Thr Phe Trp Asn Il #e Thr Leu Glu Asp Glu            100       #           105       #           110Gly Cys Tyr Met Cys Leu Phe Asn Thr Phe Gl #y Phe Gly Lys Ile Ser        115           #       120           #       125Gly Thr Ala Cys Leu Thr Val Tyr Val Gln Pr #o Ile Val Ser Leu His    130               #   135               #   140Tyr Lys Phe Ser Glu Asp His Leu Asn Ile Th #r Cys Ser Ala Thr Ala145                 1 #50                 1 #55                 1 #60Arg Pro Ala Pro Met Val Phe Trp Lys Val Pr #o Arg Ser Gly Ile Glu                165   #               170   #               175Asn Ser Thr Val Thr Leu Ser His Pro Asn Gl #y Thr Thr Ser Val Thr            180       #           185       #           190Ser Ile Leu His Ile Lys Asp Pro Lys Asn Gl #n Val Gly Lys Glu Val        195           #       200           #       205Ile Cys Gln Val Leu His Leu Gly Thr Val Th #r Asp Phe Lys Gln Thr    210               #   215               #   220Val Asn Lys Gly Tyr Trp Phe Ser Val Pro Le #u Leu Leu Ser Ile Val225                 2 #30                 2 #35                 2 #40Ser Leu Val Ile Leu Leu Val Leu Ile Ser Il #e Leu Leu Tyr Trp Lys                245   #               250   #               255Arg His Arg Asn Gln Asp Arg Gly Glu Leu Se #r Gln Gly Val Gln Lys            260       #           265       #           270 Met Thr<210> SEQ ID NO 20 <211> LENGTH: 837 <212> TYPE: DNA<213> ORGANISM: Rattus norvegicus <400> SEQUENCE: 20atgggcagtc cggtattcag gagacctttc tgccatctgt ccacctacag cc#tgctctgg     60gccatagcag cagtagcgct gagcacagct caagtggaag tggtgaccca gg#atgaaaga    120aagctgctgc acacaactgc atccttacgc tgttctctaa aaacaaccca gg#aacccttg    180attgtgacat ggcagaaaaa gaaagccgta ggcccagaaa acatggtcac tt#acagcaaa    240gcccatgggg ttgtcattca gcccacctac aaagacagga taaacatcac tg#agctggga    300ctcttgaaca caagcatcac cttctggaac acaaccctgg atgatgaggg tt#gctacatg    360tgtctcttca acatgtttgg atctgggaag gtctctggga cagcttgcct ta#ctctctat    420gtacagccca tagtacacct tcactacaac tattttgaag accacctaaa ca#tcacgtgc    480tctgcaactg cccgcccagc ccctgccatc tcctggaagg gcactgggtc ag#gaattgag    540aatagtactg agagtcactc ccattcaaat gggactacat ctgtcaccag ca#tcctccgg    600gtcaaagacc ccaaaactca ggttggaaag gaagtgatct gccaggtttt at#acttgggg    660aatgtgattg actacaagca gagtctggac aaaggatttt ggttttcagt cc#cactgctg    720ctgagcattg tttctctggt aattcttctg gtcttgatct ccatcttatt at#actggaaa    780cggcaccgaa atcaggagcg gggtgagtca tcacagggga tgcaaagaat ga#aataa       837 <210> SEQ ID NO 21 <211> LENGTH: 278 <212> TYPE: PRT<213> ORGANISM: Rattus norvegicus <400> SEQUENCE: 21Met Gly Ser Pro Val Phe Arg Arg Pro Phe Cy #s His Leu Ser Thr Tyr1               5    #                10   #                15Ser Leu Leu Trp Ala Ile Ala Ala Val Ala Le #u Ser Thr Ala Gln Val            20       #            25       #            30Glu Val Val Thr Gln Asp Glu Arg Lys Leu Le #u His Thr Thr Ala Ser        35           #        40           #        45Leu Arg Cys Ser Leu Lys Thr Thr Gln Glu Pr #o Leu Ile Val Thr Trp    50               #    55               #    60Gln Lys Lys Lys Ala Val Gly Pro Glu Asn Me #t Val Thr Tyr Ser Lys65                   #70                   #75                   #80Ala His Gly Val Val Ile Gln Pro Thr Tyr Ly #s Asp Arg Ile Asn Ile                85   #                90   #                95Thr Glu Leu Gly Leu Leu Asn Thr Ser Ile Th #r Phe Trp Asn Thr Thr            100       #           105       #           110Leu Asp Asp Gly Gly Cys Tyr Met Cys Leu Ph #e Asn Met Phe Gly Ser        115           #       120           #       125Gly Lys Val Ser Gly Thr Ala Cys Leu Thr Le #u Tyr Val Gln Pro Ile    130               #   135               #   140Val His Leu His Tyr Asn Tyr Phe Glu His Hi #s Leu Asn Ile Thr Cys145                 1 #50                 1 #55                 1 #60Ser Ala Thr Ala Arg Pro Ala Pro Ala Ile Se #r Trp Lys Gly Thr Gly                165   #               170   #               175Ser Gly Ile Glu Asn Ser Thr Glu Ser His Se #r His Ser Asn Gly Thr            180       #           185       #           190Thr Ser Val Thr Ser Ile Leu Arg Val Lys As #p Pro Lys Thr Gln Val        195           #       200           #       205Gly Lys Glu Val Ile Cys Gln Val Leu Tyr Le #u Gly Asn Val Ile Asp    210               #   215               #   220Tyr Lys Gln Ser Leu Asp Lys Gly Phe Trp Ph #e Ser Val Pro Leu Leu225                 2 #30                 2 #35                 2 #40Leu Ser Ile Val Ser Leu Val Ile Leu Leu Va #l Leu Ile Ser Ile Leu                245   #               250   #               255Leu Tyr Trp Lys Arg His Arg Asn Gln Glu Ar #g Gly Glu Ser Ser Gln            260       #           265       #           270Gly Met Gln Arg Met Lys         275 <210> SEQ ID NO 22 <211> LENGTH: 837<212> TYPE: DNA <213> ORGANISM: Mus musculus <400> SEQUENCE: 22atgggcagtc tggtattcag gagacctttc tgccatctct ccacctacag cc#tgatttgg     60ggcatagcag cagtagcgct gagcacagct caagtggaag tggtgaccca gg#atgaaaga    120aaggcgctgc acacaactgc atccttacga tgttctctaa aaacatccca gg#aacccttg    180attgtgacat ggcagaaaaa gaaagccgtg agcccagaaa acatggtcac ct#acagcaaa    240acccatgggg ttgtaatcca gcctgcctac aaagacagga taaatgtcac ag#agctggga    300ctctggaact caagcatcac cttctggaac acacacattg gagatggagg ct#gctacatg    360tgtctcttca acacgtttgg ttctcagaag gtctcaggaa cagcttgcct ta#ctctctat    420gtacagccca tagtacacct tcactacaac tattttgaac accacctaaa ca#tcacttgc    480tctgcgactg cccgtccagc ccctgccatc acctggaagg gtactgggac ag#gaattgag    540aatagtaccg agagtcactt ccattcaaat gggactacat ctgtcaccag ca#tcctccgg    600gtcaaagacc ccaaaactca agttgggaag gaagtgatct gccaggtttt at#acctgggg    660aatgtgattg actacaagca gagtctggac aaaggatttt ggttttcagt tc#cactgttg    720ctaagcattg tttctctggt aattcttctg atcttgatct ccatcttact at#actggaaa    780cgtcaccgaa atcaggagcg gggtgaatca tcacagggga tgcaaagaat ga#aataa       837

We claim:
 1. A method of suppressing an immune response comprisingadministering an effective amount of an OX-2 protein to an animal inneed thereof, wherein said OX-2 protein is capable of suppressing animmune response.
 2. A method according to claim 1 wherein the OX-2protein comprises the amino acid sequence shown in SEQ ID NO:2, SEQ IDNO:19 or SEQ ID NO:21.
 3. A method according to claim 1 wherein saidOX-2 protein can inhibit an in vitro immune response selected from thegroup consisting of: inhibiting a mixed leukocyte reaction; inhibiting acytotoxic T lymphocyte response; inhibiting interleukin-2 production;and inhibiting interferon-γ production.
 4. A method according to claim 1wherein the OX-2 protein is a soluble fusion protein.
 5. A methodaccording to claim 4 wherein the soluble fusion protein comprises anOX-2 protein linked to an immunoglobulin Fc region.
 6. A methodaccording to claim 5 wherein the soluble fusion protein comprises OX-2ED-Fc.
 7. A method according to claim 1 wherein the OX-2 protein is froma human OX-2 protein.
 8. A method of suppressing an immune responsecomprising administering an effective amount of a fragment of an OX-2protein to an animal in need thereof, wherein said OX-2 protein fragmentis capable of suppressing an immune response selected from the groupconsisting of: inhibiting a mixed leukocyte reaction; inhibiting acytotoxic T lymphocyte response; inhibiting interleukin-2 production;and inhibiting interferon-γ production.
 9. A method according to claim 8wherein the OX-2 protein fragment comprises a fragment of an amino acidsequence shown in SEQ ID NO:2, SEQ ID NO:19 or SEQ ID NO:21.
 10. Amethod according to claim 8 wherein the OX-2 protein fragment is asoluble fusion protein.
 11. A method according to claim 10 wherein thesoluble fusion protein comprises an OX-2 protein fragment linked to animmunoglobulin Fc region.
 12. A method according to claim 11 wherein thesoluble fusion protein comprises OX-2 ED-Fc.
 13. A method according toclaim 8 wherein the OX-2 protein fragment is from a human OX-2 protein.